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Depression constitutes a spectra of symptoms that adversely affect an individual’s cognitive, emotional, motivational, and physiological well-being; this collection of heterogenous symptoms and pathologies is termed major depressive disorder (MDD), and for as many as 40% of individuals suffering from MDD, medications are not able to provide sufficient and/or long-lasting therapeutic relief^1,2. Functional imaging studies and neuropathological studies with post-mortem human brains implicate dysfunction in the nucleus accumbens (NAc) and ventral tegmental area (VTA), critical nuclei in the brain’s dopaminergic (DAergic) reward pathways^2-6. The VTA consists of a major population of DA neurons which primarily innervates the NAc^2,5. Dysfunctional neurophysiology and plasticity in these nuclei likely contribute to some of the symptoms of MDD, specifically loss of pleasure and motivation (anhedonia)^2-6. Anhedonia is studied using chronic social defeat stress (CSDS) which elicits depression-associated behaviors (e.g., reduced social interactions, increased anhedonia, negative body weight changes) in 70% of mice (i.e., termed stress-susceptible) while the remaining 30% do not undergo these adverse behavioral changes (i.e., termed stress-resilient)^2,7.

The structure and function of DAergic neurons in VTA and the DA receptor 1 (D1R)- and D2R-expressing NAc neurons are remodeled during CSDS^2,8-16. Stress-induced anhedonia to model depression-like behavior results in reduced spontaneous excitatory inputs to D1R-expressing MSNs, whereas D2R-expressing MSNs undergo the opposite change^17,18. D1R-expressing (but not D2R) accumbal neurons also display reduced dendritic arborization (morphological plasticity), an important parameter in excitatory neurotransmission for the MSNs as dendritic spines are the primary site of excitatory synapses^{8,10-12,19,20}.

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Actin PTM Background

Actin is a well-characterized, abundant, and essential cytoskeletal protein. Its dynamic properties allow it to shift between monomeric (G-actin) and polymeric (F-actin) states, which is vital for many cellular processes.  Actin’s dynamicity and function is regulated by many internal and external cues that are facilitated by actin binding proteins (ABPs), signal transducers, and others.  Additionally, several studies now indicate that actin itself is highly modified by post-translational modifications (PTMs); furthermore, intensive studies of specific actin PTMs have detailed their effect on actin dynamics, ABP interactions, and actin-dependent physiology^1,2. For example, actin N-terminal acetylation, lysine acetylation, arginylation, SUMOylation, and ubiquitination have been studied.  Here, current studies on physiologic oxidation of actin at methionine (Met)44 and Met47 are summarized.

Actin Oxidation: Focus on Methionine Sulfoxide (MetO)

The oxidation field in general has focused on pathological oxidation (oxidative stress) induced by naturally formed oxidants like H₂O₂, and while it was initially hypothesized that oxidation was “toxic”, it is now known that oxidation is a signaling mechanism for both pathological and physiological events³.  Most of this work related to reactive oxygen species (ROS) focused on thiol-based cysteine (Cys) modifications. This is also true in the case of actin, whereby, most of the early work on actin oxidation utilized H₂O₂ treatment and resulted in altered F-actin content and polymerization activity as defined by enhanced lag time, slower polymerization rate, and lower polymerization extent^4-6. Further investigation showed that H₂O₂-induced oxidation of actin initially targeted Cys374, but also targeted several Mets, including, Met44, Met47, Met176, Met190, Met269, and Met355 in vitro⁷. MetO oxidation happens in vivo under normal or stress conditions; however, Manta and Gladsyshev suggested that ROS-induced MetO formation in vivo occurs very inefficiently relative to the kinetics of reducing enzymes like MsrA⁸. Thus, the question of whether actin’s methionines can become oxidized enzymatically in vivo still remained unanswered. A seminal study by the Terman group identified a role for the enzyme, MICAL (molecule interacting with CasL), in mediating oxidation of Met44 and Met47 of actin in vitro⁹. These in vitro studies showed that MetO at Met44 was sufficient to promote both severing of filaments and decreased polymerization. Additionally, mis-regulation of actin MetO produced profound morphological consequences as overexpression of MICAL in Drosophila resulted in deformed bristle formation but was rescued with M44L actin mutants; thereby, defining its role in vivo^9,10. A recent study identified a physiologic role for MICAL-1 oxidation of F-actin by determining that this mechanism is critical for depolymerizing actin during the terminal steps of cytokinesis¹¹. These studies shed light on the physiologic importance that actin MetO has on actin’s molecular, cellular, and morphologic functions. 

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Microglia are the primary immune cells, the so-called professional phagocytes, of the mammalian brain. Microglia continually scan the entire brain in their resting state and when neurotoxic pathogens and damaged cellular machinery are detected, they are activated to eliminate them^1-2 (Fig. 1). In healthy neurons, microglia also contribute to essential cellular functions such as neurogenesis, neurodevelopment, and neural plasticity^3-4. Of similar importance are the roles microglia have in neurodegenerative diseases characterized by aggregates of pathogenic, misfolded proteins such as Parkinson’s disease (PD; Lewy bodies) and Alzheimer’s disease (AD; Aβ plaques). Microglia-mediated neuroinflammation is a common pathophysiology in PD and AD human brains, as well as in in vivo animal model brains ^1-2,4-9. Moreover, recent genetic and transcriptomic studies revealed microglia-associated signaling cascades as critical players in AD pathogenesis^1-7. This newsletter discusses microglia activation and neuroinflammation in PD and AD.

PD is characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta, a corresponding loss of dopaminergic (DAergic) terminals throughout the basal ganglia, and Lewy bodies which are composed primarily of intracellular α-synuclein aggregates. Lewy bodies are a pathophysiological hallmark of PD and trigger microglia-mediated neuroinflammation, which itself is another neuropathological correlate of PD^1,2,8,10 (Fig. 1). In the brains of PD patients, Lewy body neurites are associated with activated microglia and α-synuclein deposits are correlated with inflammatory markers. In vitro cell culture and in vivo animal models also demonstrate a relationship between α-synuclein aggregates and microglial activation^2,8,10. 

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Schematic of Atherogensis regulation through epsin’s ubiquitin-dependent interaction with LRP-1 receptors

In a recent study, Dr. Chen and colleagues identified a critical role of myeloid epsins to promote atherogenesis through ubiquitin-dependent regulation of the LRP-1 receptor.  They utilized elegant, myeloid-specific epsin double-knockout mouse models to define its role in atherogenesis progression and showed that epsin loss decreased atherosclerotic lesions.  They investigated this pathological phenomenon at the cellular level and identified critical changes in macrophages, defined by suppression of a pro-inflammatory phenotype and enhancement of an anti-inflammatory phenotype.  Further examination led to the molecular discovery that levels of LRP-1, a surface receptor that promotes an anti-inflammatory phenotype, were enhanced in the absence of myeloid epsins.  Importantly, they discovered that LRP-1 and epsins interact through epsin’s ubiquitin-interacting motif.  In response to oxidizied-LDL, a pro-inflammatory stimulant, LRP-1’s ubiquitination increased, which enhanced its interaction with epsins.  Cytoskeleton’s ubiquitin antibody (Cat. # AUB01) was a critical reagent that was utilized to investigate the ubiquitination state of LRP-1, which provided a key finding in unraveling the mechanistic interaction and regulation of these proteins in atherogenesis.   This study provides the foundation to target myeloid-specific epsins as a potential therapeutic to treat atherogenesis, and it will be interesting to see if they can regulate epsin through targeting its ubiquitin-interacting motif.

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Brophy ML et. al., Myeloid-Specific Deletion of Epsins 1 and 2 Reduces Atherosclerosis by Preventing LRP-1 Downregulation. 2019 Feb 15;124(4):e6-e19. doi: 10.1161/CIRCRESAHA.118.313028

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Click Here to View Introducing the New 2019 Cytoskeleton Minicatalog! GEF and K-Ras4B Mutated Proteins New products = new discoveries! Signal-Seeker™ Kits New comprehensive kits! Signal-Seeker™ Antibodies Acetyl-Lysine, Ubiquitin, and SUMO2/3!

Renewable Energy and Recycling at Cytoskeleton, Inc.

In 2019, Cytoskeleton made the move to power its sole facility at Denver, Colorado, USA with 100% renewable energy (wind and solar). The local electric company (Xcel energy) has enabled this by its Colorado State mandate to produce 30% of its electricity from wind and solar by 2020 (and 100% by 2040). We are proud to support the 2016 Paris Agreement on Climate Change with this change.

In addition, late in 2018 a recycling program was started at Cytoskeleton. To date we achieve 75% recycled waste, while looking for more ways to recycle the remainder. More changes will take place to utilize post-consumer materials wherever possible.

We look forward to a peaceful, efficient and productive scientific culture in 2019 and beyond.

The Team at Cytoskeleton, Inc.

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Studying the causes of human central nervous system (CNS) diseases and disorders and developing impactful therapies requires in vitro and in vivo disease models that faithfully recapitulate the respective neuropathophysiology while also supporting the neurons with the necessary cellular machinery to respond to therapies in a manner that offers translational results. Moreover, it is imperative to study the earliest neuropathophysiological changes as these provide the best opportunity for early diagnosis and intervention. Changes in the structure and/or function of neuronal synapses are often the earliest pathological changes in many neurodegenerative diseases and CNS disorders. To detect early synaptic dysfunction, scientists utilize live cell imaging which provides single cell resolution with real-time analysis of changing experimental conditions. This newsletter discusses the use of live cell imaging in in vitro and in vivo studies of CNS diseases and disorders.

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Domain structures of wild-type and mutant amyloid precursor proteins.

Luu et al. recently studied the regulation of neurite (i.e., neuronal axons and dendrites) outgrowth by amyloid precursor protein (APP) homodimers in SH-SY5Y human neuroblastoma cells over-expressing either wild-type (WT) or mutant L17C APP. APP is a transmembrane glycoprotein highly expressed in the brain and spinal cord, and its metabolites are involved in various physiological and pathological functions. For the present study, the authors utilized mutant L17C APP which correlates with a 30% increase in APP homodimerization. In differentiated SH-SY5Y cells displaying a neuronal phenotype, over-expression of the mutant, but not WT, APP reduced neurite outgrowth. The mutant APP-mediated inhibition correlated with increased RhoA activity, a known negative regulator of neurite outgrowth. The mutant APP/RhoA-mediated decrease was rescued with application of either the Rho inhibitor Y27632 or conditioned media from cells over-expressing WT APP. In addition, over-expression of the microRNA miR-34a rescued the mutant phenotype concomitant with a decrease in RhoA activity. Cytoskeleton’s RhoA G-LISA activation assay Kit (Cat. # BK124) was an essential reagent in this study, providing an economical, sensitive, and reliable means to study the mechanistic role of RhoA in APP-mediated regulation of neuronal differentiation and morphogenesis, processes integral in neurodevelopment.

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Luu L. et al. 2018. Amyloid precursor protein dimerization reduces neurite outgrowth. Mol. Neurobiol. doi: 10.1007/s12035-018-1070-4.

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Crystal structure of human RCC2 as determined by X-ray diffraction. RCC2 and sulfate ion complex. PDB ID: 5GWN.

Wu et al. recently studied how over-expression or knockdown of regulator of chromosome condensation 2 (RCC2; also known as TD-60) protein affects the ability of nine chemotherapeutic drugs to induce apoptosis in three different in vitro cancer cell culture models of lung and ovarian tumors. RCC2 over-expression antagonized spontaneous and staurosporine (STS)-induced apoptosis, whereas RCC2 knockdown had the opposite effect. Besides apoptosis, cell proliferation and signaling of Rho-family GTPases (RhoA, Rac1, and Cdc42) were also evaluated since RCC2 inhibits GEF-mediated activation of Rac1. The authors found that the decrease in apoptosis in cells over-expressing RCC2 coincides with decreased Rac1 activation. Further experiments demonstrated that inhibition of Rac1 activation by over-expressed RCC2 is necessary for the development of a cell’s resistance to chemotherapeutic-induced apoptosis. Neither RhoA nor Cdc42 activation changed. Cytoskeleton’s RhoA, Rac1, and Cdc42 Activation Assay Combo Biochem Kit (Cat. # BK030) was an essential reagent in this study, providing an economical, sensitive, and reliable means to study the mechanistic role of three Rho-family GTPases in anti-cancer therapeutics. Moreover, this study may offer a screening tool for evaluating a tumor’s likely response to different chemotherapies since cells over-expressing RCC2 resisted many of the chemotherapies tested in this study.

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Wu N. et al. 2018. RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation. BMC Cancer. 18, 67.

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RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) (Cat. # BK030)

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Mechanotransduction is a multi-step biological process by which cells sense, interpret, and respond to mechanical (i.e., physical) force through conversion to biochemical signals that elicit specific cellular responses. The responses are often mechanical in nature as they involve force generation to produce cellular protrusions and retractions which require remodeling of the actin cytoskeleton, consisting of monomeric (globular; G-) and helical polymeric (filamentous; F-) actin and actin binding proteins (ABPs). ABPs dynamically organize F-actin into many different structural forms such as lamellipodia, stress fibers, filopodia, podosomes, actin asters, vortices, and stars. These different architectures serve specialized roles in the cell’s multiplex response to mechanical stimulation. A primary means by which F-actin transduces these signals is through its connections to focal adhesions and adherens junctions, which coordinate contact between the cell’s actin cytoskeleton and either the extracellular matrix or another cell, respectively(Fig. 1). Understanding the actin cytoskeleton’s role in mechanotransduction goes beyond the basic biology underlying force-induced changes in actin-based cellular structures and functions. Diseases resulting from expression of mutant ABPs render cells unable to respond to mechanical forces physiologically. In this newsletter, the role of the actin cytoskeleton in mechanotransduction is discussed.

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Schematic of phagocytic cell engulfing an apoptotic cell as mediated by recognition of phosphatidylserine (PS) signals.

Ismail et al. characterized Tctex-1, a novel binding partner of kidney injury molecule-1 (KIM-1), and described the role both proteins have in efferocytosis, the phagocytosis of apoptotic cells. Efferocytosis is an important function in tissue repair, reducing inflammation and abnormal autoimmune responses. KIM-1 is a phosphatidylserine (PS) receptor and PS is a well-known identifier of apoptotic cells. The physical and functional interactions between Tctex-1 and KIM-1 were examined in proximal tubular epithelial cells (PTECs) which can act as amateur phagocytes. A previously unknown role for Tctex-1 in KIM-1-mediated efferocytosis in PTECs was described. Efferocytosis requires re-organization of the actin and microtubule networks, prompting the authors to examine actin cytoskeletal dynamics and the potential role of RhoA and Rac1 in efferocytosis mediated by Tctex-1/KIM-1. Notably, neither RhoA nor Rac1 activities were regulated by Tctex-1 during KIM-1-dependent efferocytosis in PTECs. These data suggested the unexpected hypothesis that non-canonical pathways not involving RhoA and Rac1 are utilized by Tctex-1. Cytoskeleton’s rhodamine-phalloidin, RhoA and Rac1 G-LISA activation assay kits, and Total RhoA ELISA (Cat. # PHDR1, BK124, BK128, BK150, respectively) were essential reagents in this study, the first to describe the physical and functional roles of Tctex-1 in KIM-1-dependent efferocytosis in kidney cells.

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Ismail O.Z. et al. 2018. Tctex-1, a novel interaction partner of Kidney Injury Molecule-1, is required for efferocytosis. J. Cell. Physiol. 233, 6877-6895.

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Total RhoA ELISA (Cat. # BK150)

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The cytoskeleton of adult neurons in the central nervous system (CNS) is composed of different structural proteins, including microtubules (MTs) and F-actin. Normal cellular functions (e.g., morphology, motility, development, transport) rely upon the dynamicity of MTs and F-actin. Additionally, cytoskeletal dysfunction underlies many CNS diseases and injuries. For instance, the cytoskeletal dynamics in the adult CNS following axonal injury are not conducive to growth cone formation, axon regeneration, and recovery of function. To understand the role of the MT and F-actin cytoskeleton in adult CNS injury recovery, and how to modulate them for axon regeneration demands an understanding of cytoskeletal dynamics following axonal injury^1,2. This newsletter discusses the roles of the MT and F-actin cytoskeleton in axon regeneration in the adult CNS.

The dogma is that axon regeneration does not occur in adult CNS neurons under physiological conditions. In contrast, damaged axons in the peripheral nervous system (PNS) are regenerated following injury.  The focus on understanding this difference centers on two prominent regeneration-associated signaling cascades in the adult CNS: 1. activation of intrinsic regeneration-associated genes [RAGs]), and 2. external cues that inhibit axon re-growth^1,2.

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Rho activation in Swiss 3T3 cells. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1) and nuclear blue DNA staining with Dapi. Cells were activated with Cat.# CN03.

Corvol et al. investigated the gene Family with sequence similarity 13 member A (FAM13A) and its contribution to the pathophysiology of cystic fibrosis (CF), a monogenic lung disease characterized by an excessive inflammatory response. Disease severity varies and is influenced by genetic modifiers. Here, the authors confirm that FAM13A is a genetic modifier of the CF lung phenotype and can impact the severity of lung disease in CF patients. To mimic inflammation that accompanies CF, lung epithelial cells and primary human bronchial epithelial cells from CF patients were treated with IL-1β and TGF-β, pro-inflammatory cytokines. Each cytokine decreased expression of FAM13A. FAM13A has a Rho GTPase activating protein (GAP) domain, prompting the authors to measure RhoA activity and associated F-actin stress fiber formation following siRNA-mediated reduction in FAM13A expression. An increase in both occurred, whereas E-cadherin expression decreased. Additionally, TGF-β stimulation of siRNA-treated cells displayed a further reduction of E-cadherin concomitant with increased expression of α-smooth muscle actin and vimentin. These changes are characteristic of an epithelial to mesenchymal transition. Cytoskeleton’s RhoA pull-down activation assay kit (Cat. # BK036) was essential in identifying a molecular pathway underlying FAM13A-mediated alterations in CF lung phenotypes.

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Corvol H. et al. 2018. FAM13A is a modifier gene of cystic fibrosis lung phenotype regulating rhoa activity, actin cytoskeleton dynamics and epithelial-mesenchymal transition. J. Cyst. Fibros. 17, 190-203.

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Neuronal polarity describes the spatial, morphological, structural, and functional differentiations that occur in neurons during early development that results in the formation of a single axon and multiple dendrites. Axons and dendrites are responsible for directional signaling in neurons - receiving, processing, and transmitting information from the postsynaptic dendrites to the axon of the postsynaptic neuron. The majority of excitatory inputs at the dendrites occur at dendritic spines. Polarization of the neuron begins with the loss of the symmetric shape of a round newborn neuron via formation of minor neurites^1-4. Neuronal polarization depends upon: 1. the polarity of microtubules (MTs), one of the primary cytoskeletal polymers in cells, and 2. polarized cargo transport by kinesins and dynein along the MTs in axons and dendrites^4,5.

MTs are intrinsically polar filaments composed of alpha/beta-tubulin heterodimers with an exposed beta-tubulin at the plus end and an exposed alpha-tubulin at the minus end^5-7. MT polarity directs: 1. location of MT assembly/disassembly; 2. where MT-associated proteins (MAPs; e.g., +TIPs, motors) bind MTs in the cell; and 3. motor-driven traffic along MTs. Importantly, MTs are integral for nearly all normal neuronal functions and MT disruption underlies several neural pathologies^7-10...

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Tubulin polymerization using the fluorescence based tubulin polymerization assay (Cat. # BK011P). Tubulin was incubated alone (Control), with Paclitaxel or Vinblastine. Each condition was tested in duplicate. Polymerization was measured by excitation at 360 nm and emission at 420 nm.  The three Phases of tubulin polymerization are marked for the control polymerization curve; I: nucleation, II: growth, III: steady state equillibrium.

Burke et al. investigated the potential of tubulysin M, a potent tubulin binder and anti-mitotic tetrapeptide, as the cytotoxic component of antibody-drug conjugates (ADCs). Structurally, the C11 acetoxy moiety within the tubuvaline residue of tubulysins is important for their high potency. However, tubulysin activity is compromised when this moiety undergoes deacetylation. Replacement of this moiety with an ether or ester group created stabilized tubulysin M analogues. Wild-type tubulysin M and two stabilized analogues, both in free drug and conjugate form, were evaluated pre-clinically as anti-cancer therapeutics in in vitro and in vivo model systems. First, in vitro cell and biochemical potencies of the free forms of these tubulysins was determined. The free and conjugated forms were further evaluated using assorted cancer cell lines and in vivo tumor xenograft models, including those with multi-drug resistant phenotypes. Cytoskeleton Inc. produced sheep brain tubulin (Cat.# CS-T234S) in a specialized buffer for use in competitive fluorescence polarization tubulin binding assays to measure the biochemical potencies of tubulysin M and analogues in free drug form. These biochemical data complemented cell potency results and buttressed the rationale for further development and testing of stabilized tubulysins as a novel payload class for ADCs in targeted anti-cancer therapies.

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Burke P.J. et al. 2018. Glucuronide-linked antibody-tubulysin conjugates display activity in MDR+ and heterogeneous tumor models. Mol. Cancer Ther. 17, 1752-1760.

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Tubulin protein plus glycerol, Sheep Brain (>99% pure) (Cat. # CS-T234S)

Immunofluorescence using Phosphotyrosine Antibody: Human epidermoid carcinoma A431 cells, untreated (3A) or treated (3B) with EGF (100 ng/ml for 3 minutes), and NIH3T3, untreated (3C) or treated (3D) with H2O2-activated sodium orthovanadate (100 µM for 10 minutes), were stained as described in the method. Phosphotyrosine and nuclei were visualized in green fluorescence and blue DAPI staining, respectively.

Kline et al. recently studied regulation of intercellular bridges in Drosophila egg chambers. Known as ring canals, the bridges transfer cytoplasmic materials between neighboring cells; in this case, from nurse cells to the oocyte over the course of oogenesis. Ring canals are essential for maintaining an organism’s fertility. Ring canal size and stability rely upon dynamic re-organization of F-actin. Here, the Ste20 family kinase misshapen (msn) has a novel role in regulating ring canal size and stability with changes in msn expression levels or localization altering the F-actin cytoskeleton. Phosphotyrosine (pTyr) signal in ring canals strongly overlaps with actin in control egg chambers. pTyr signal was used as a read-out to measure msn localization and activity in control vs msn-RNAi-treated egg chambers. In the latter, pTyr localized to nurse cell membranes and most ring canals. However, pTyr fluorescence in ring canals was variable, even within the same egg chamber, and did not overlap with actin signal. Hence, msn is not necessary for recruiting actin to the ring canal, but is essential for maintaining a ring canal’s actin-based structure. Cytoskeleton’s anti-phosphotyrosine antibody (Cat.# APY03; clone 11G2) was essential in the subcellular localization of pTyr signal in control and msn-depleted ring canals.

Link to citation:

Kline A. et al. 2018. The misshapen kinase regulates the size and stability of the germline ring canals in the Drosophila egg chamber. Dev. Biol. 440, 99-112.

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Rab GTPases, members of the Ras super-family of GTPases, express at least 60 isoforms in humans¹ with 24 enriched in, or specific for, the central nervous system (CNS)². Rab GTPases are  integral regulators of intracellular membrane trafficking, regulating the formation, maturation, transport, tethering, and fusion of vesicles in the endomembrane system³. In neurons, Rab-mediated membrane/vesicle trafficking is involved in virtually every aspect of neuron physiology, and dysfunction has been implicated in several neurodegenerative diseases^2,4-7 (Fig. 1). Indeed, dysfunctional membrane trafficking is an early marker of neurodegeneration⁵. Here, Rab-mediated trafficking defects in Parkinson’s disease (PD) and Alzheimer’s disease (AD) are reviewed.

The cause of PD is primarily idiopathic – only a small fraction of PD cases are familial and attributable to mutations in proteins that also are involved in Rab activity and membrane trafficking. Mutant proteins include Rab39b, α-synuclein, PTEN-induced putative kinase (PINK1), and leucine-rich repeat kinase 2 (LRRK2)^4,6,7. Rab39b is particularly interesting because loss-of-function mutations in this gene are directly linked to inherited early-onset PD with Lewy body pathology and also...

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Neutrophil, a class of leukocytes, with segmented nucleus and intracellular granules; 2D rendering.

Activation of cytokine tumor necrosis factor (TNF)-mediated molecular pathways induces changes in the morphology, motility, and adhesion of neutrophils, a class of leukocytes, early in vascular inflammation and in related diseases, including atherosclerosis, sickle cell anemia, stroke, and sepsis. Neutrophils migrate to sites of inflammation where they adhere to the vascular endothelium. Alterations in a cell’s shape, motile behavior, and/or adhesiveness, require dynamic re-organization of the actin cytoskeleton. Here, Silveira et al. report that actin polymerization was necessary for TNF’s effects, which in turn, focused attention on GTPases such as RhoA, a well-known regulator of actin cytoskeletal dynamics and F-actin re-modeling. As might be expected, TNF increased RhoA activity in neutrophils, but through RhoA-mediated pathways independent of the downstream effector protein Rho kinase. Instead, mDia, a regulator of actin nucleation and member of the formin family of Rho effector proteins, was implicated. SMIFH2, a formin inhibitor, eliminated TNF-mediated increases in neutrophil morphological changes and adhesion. Cytoskeleton’s RhoA G-LISA activation assay kit (Cat. # BK124) was essential in quantifying RhoA activity in a sensitive and reliable manner. Deciphering the molecular pathways responsible for TNF-stimulated changes in neutrophil morphology, motility, and adhesion offers a therapeutic target for reducing leukocyte-regulated inflammatory responses.

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Silveira A.A.A. et al. 2018. TNF induces neutrophil adhesion via formin-dependent cytoskeletal reorganization and activation of beta-integrin function. J. Leukoc. Biol. 103, 87-98.

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RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays (Cat. # BK124)

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In mammalian cells, the small ubiquitin-like modifier (SUMO) family contains four isoforms (SUMO1, SUMO2, SUMO3, and SUMO4). SUMO2 and SUMO3 are almost identical, with only a difference in three amino acid residues. SUMO1 shares 48% identity with SUMO2/3¹. SUMO4 is about 85% identical to SUMO2/3, but it is unclear whether SUMO4 can be conjugated to substrates². Similar to ubiquitination, SUMOylation requires a three enzymes system (E1, E2, and E3) to conjugate SUMO covalently to target substrates. Briefly, SUMO is first activated by the SUMO E1 activating heterodimeric enzyme SAE1/SAE2 by adenylation in an ATP-dependent reaction. The activated SUMO is then transferred to the SUMO E2 conjugating enzyme UBC9 and finally conjugated to a target protein by a SUMO E3 ligase (e.g., PIAS family members, Ran binding protein 2). The covalently linked SUMO can be removed by sentrin-specific proteases (SENPs), a process known as deSUMOylation³ (Fig. 1). SUMOylation is an essential post-translational modification (PTM) that regulates the activity, subcellular localization, stability, and functions of target proteins and thereby modulates almost all major cellular pathways⁴. Therefore, it is not surprising that many diseases are associated with dysregulation of SUMOylation. In this newsletter, the roles of SUMOylation/deSUMOylation in cancer are discussed.

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Visualization of acetylated mitochondrial proteins in Swiss 3T3 cells. Acetylated mitochondrial-localized proteins were labeled with acetyl-lysine antibody (Cat. # AAC02) and alexa-488 secondary (green), or a fluorescent mitochondrial marker (mitotracker orange) shown in red.

Post-translational modification (PTM) crosstalk is recognized as a major cell-regulatory mechanism; a theory validated by in-depth, functional analysis of several well-investigated proteins. Recently, Horita et al. examined the PTM crosstalk that occurs in acetylated mitochondrial proteins in response to a mitochondrial stress-inducing agent, hydrogen peroxide (H₂O₂). Visual changes in the acetylated state of mitochondrial proteins were investigated by immunofluorescence and showed dynamic changes in response to H₂O₂. These investigators validated the acetylation state of 10 mitochondrial targets and also measured endogenous changes to the acetylation state of these proteins using Signal-Seeker acetyl-lysine detection tools. The endogenous acetylation (Ac), ubiquitination (Ub), SUMOylation 2/3 (SUMO 2/3), and tyrosine phosphorylation (pY) state of four target mitochondrial proteins were investigated with these toolkits to examine if PTM crosstalk commonly occurs on mitochondrial proteins. Each of the four proteins had unique PTM profiles, but diverging acetylation and ubiquitin or SUMO 2/3 signals appeared to be a common theme. Cytoskeleton’s pY, Ub, SUMO 2/3, and Ac Signal-Seeker PTM detection kits (Cat. # BK160, BK161, BK162, and BK163 respectively) were essential tools used in this study to examine endogenous and dynamic PTM crosstalk.

Link to citation:

Horita H. et al. 2018. Utilizing Optimized Tools to Investigate PTM Crosstalk: Identifying Potential PTM Crosstalk of Acetylated Mitochondrial Proteins. Proteomes. Doi: 10.3390/proteomes6020024.

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Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)

Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ Acetyl-Lysine Detection Kit (Cat. # BK163)

BlastR Rapid Lysate Prep Kit (Cat. # BLR01)

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Ras GTPases regulate cell proliferation pathways, making them important molecules in oncogenesis and cancer cell migration and invasion. The four isoforms of Ras, H-Ras, N-Ras, K-Ras4A, and K-Ras4B (due to alternative splicing), were identified over 30 years ago for their oncogenic activation in human tumors. Activating Ras mutations are single amino acid substitutions (e.g., G12C, G12V, G12D) and have been identified in approximately 30% of all human cancers.

The same signaling pathways activate all Ras isoforms via guanine exchange factor (GEF)-mediated exchange of GDP for GTP, followed by binding to the same effector proteins. However, Ras oncogenic isoforms are differentially expressed aacross different cancers with oncogenic specificity significantly favoring K-Ras,. Indeed, K-Ras is the most common mutated Ras isoform (86% of all Ras mutations) and is correlated with over 21% of human cancers. In particular, K-Ras is the predominant or exclusive Ras gene mutated in three of the top four cancers with the highest mortality rates in the US: lung, colon, and pancreatic cancers. In most instances, the K-Ras4B is the primary isoform mutated in K-Ras-associated cancers. This newsletter discusses potential explanations for the biological basis of K-Ras’s oncogenic specificity.

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Scientists at Cytoskeleton Inc. recently investigated post-translational modifications (PTMs) that regulate the Rho GTPase family of proteins. Here, the authors characterized the functional regulation of RhoGDIα by SUMOylation 2/3 (SUMO 2/3) and acetyl-lysine PTMs. These studies led to the identification of endogenous SUMO 2/3 and acetylation (Ac) modifications of RhoGDIα. Interestingly, the microtubule stabilizer paclitaxel induced both Ac and SUMO 2/3 of RhoGDIa in a time-dependent fashion.  These changes in the PTM state of RhoGDIα altered the protein’s ability to inhibit RhoA activity. Experiments performed with the HDAC inhibitor, TSA, provided evidence of crosstalk between RhoGDIa SUMO 2/3 and Ac in a cell-type dependent fashion and further support the hypothesis that these PTMs may alter RhoGDIa’s function. Cytoskeleton's SUMO 2/3, and Ac Signal-Seeker™ PTM detection kits (Cat. # BK162, and BK163, respectively) were essential reagents in this study, providing a novel toolset for simple and effective investigation of established and novel PTMs for any target protein.

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Horita H et al. Paclitaxel induces post-translational modifications of RhoGDI alpha: a potential mechanism to regulate RhoA activity. Mol. Biol. Cell. ASCB Annual Meeting 2017, Poster B624.

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Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)

Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (Cat. # BK162)

Signal-Seeker™ Acetyl-Lysine Detection Kit (Cat. # BK163)

BlastR Rapid Lysate Prep Kit (Cat. # BLR01)

Paclitaxel (Taxol) (Cat. # TXD01)

Acti-stain^™ 488 phalloidin (Cat. # PHDG1)

RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays (Cat. # BK124)

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The ubiquitin-proteasome system (UPS) is a well-characterized protein degradation system in cells whose dysfunction is implicated in many diseases, including neurodegeneration and cancer. Major UPS components are ubiquitin (Ub), Ub ligases, Ub hydrolases (deubiquitinases [DUBs]), and the proteasome. Activation of the UPS begins with attachment of an 8 kDa ubiquitin protein to a target protein by a three step cascade carried out by Ub ligases. Ub itself can be ubiquitinated, leading to poly-ubiquitination, a marker for proteasomal recognition and ultimately degradation (Fig. 1). This is an oversimplification as there are several unique Ub specific chains, as well as mono-ubiquitination, that can regulate multiple facets of a protein’s function. Due to the prevalence of UPS dysfunction in disease and an increased molecular understanding of how ubiquitination degrades protein, interest in targeting the UPS for therapeutic intervention has grown substantially.

Given the proteasome’s role in regulated degradation of poly-ubiquitinated proteins and its dysfunction in cancer, researchers posited that inhibition of the proteasome may be effective for treating cancer cachexia (wasting syndrome). This hypothesis spurred the development of early proteasome inhibitors like MG-132. MG-132’s pharmacological properties precluded its use clinically; however, it launched the...

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Actin cytoskeleton morphological changes induced by Rho/Rac/Cdc42 Activator I (Cat. # CN04) treatment of Swiss 3T3 cells.  Cells were fixed, stained with Acti-stain™ 488 phalloidin (Cat. # PHDG1), and visualized by fluorescence microscopy.

Kim et al. recently evaluated the potential anti-tumor effects of aripiprazole (ARP), an atypical anti-psychotic used to treat neuropsychiatric disorders. In the search for novel anti-cancer therapeutics, the authors examined if ARP affects the viability and migration of various cancer cell lines (e.g., U251 glioma cells, MDA-MB-231, HEK293) by a variety of in vitro and in vivo experiments. ARP inhibited cancer cell growth, survival, and motility by inducing apoptosis. Next, possible molecular targets of ARP were explored. Many anti-cancer compounds target the oncogenic tyrosine kinase Src as it is important for cancer cell survival, proliferation, migration, and invasion. ARP inhibited phosphorylation of Src, its auto-phosphorylation activity, and Src’s kinase activity. To decipher how ARP affects Src, ARP’s effects on actin polymerization were examined as re-arrangement of the actin cytoskeleton activates Src. In conjunction with phalloidin staining images, the polymerization data demonstrated that the actin cytoskeletal dynamics were not molecular targets of ARP-mediated cancer cell apoptosis. Cytoskeleton’s actin polymerization assay kit (Cat.# BK003) was essential in demonstrating that ARP does not carry out its Src-mediated anti-tumor effects through an interaction with actin and regulation of actin dynamics, thus eliminating one molecular substrate in the search for ARP’s mechanism of anti-cancer actions.

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Kim M.S. et al. 2018. Src is the primary target of aripiprazole, an atypical antipsychotic drug, in its anti-tumor action. Oncotarget. 9, 5979-5992.

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Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin (Cat. # BK003)

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Dendritic spines are the post-synaptic component of most excitatory glutamatergic synapses and primary site of synaptic structural plasticity for modulating synaptic function. Activity-dependent structural plasticity in spines (i.e., spine morphogenesis) depends upon dynamic re-organization of F-actin, the primary structural component of spines. Spine morphogenesis is important for normal learning and memory and the development of neurodegenerative diseases and neurological disorders.

The RhoA, Rac1, and Cdc42 GTPases regulate spine morphogenesis; RhoA inhibits spine growth and stability, whereas Rac1 and Cdc42 exert the opposite effect. In reality, Rho family regulation of spine structural plasticity is much more complex. Precise spatiotemporal regulation of Rho GTPases is with guanine exchange factors (GEFs) triggering  GTP/GDP exchange and GTPase activating proteins (GAPs) stimulating intrinsic GTPase activity. At least eight Rho family GEFs regulate spine morphogenesis and these GEFs are activated through a variety of receptor signaling pathways, including glutamatergic NMDA receptors (NMDARs) and receptor tyrosine kinases (RTKs). NMDARs mediate calcium influx and subsequent activation of calcium/calmodulin-dependent kinases (CaMKs), which phosphorylate Rho family GEFs, essential for GEF activity. In this newsletter, the regulation of spine structural plasticity by the Rho family GEFs Kalirin7 (Kal7; the most abundant isoform in adult brain), Trio-9 (the most abundant isoform in hippocampus), Tiam1, RasGRF2, DOCK10, DOCK180, ephrexin1, and ephrexin5 is discussed.

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Protein post-translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, and SUMOylation, to name but a few, have evolved to diversify the functions of a single protein and account for the vast increase in proteome complexity and functional diversity. A prime example of the complex and dynamic regulatory power PTMs confer is the Wnt/β-catenin signaling pathway. This pathway regulates cellular proliferation, differentiation, and migration during embryonic development and adult cell homeostasis. In addition, dysregulation of Wnt/β-catenin signaling is implicated in multiple pathological conditions, including carcinogenesis and degenerative diseases. In canonical Wnt-mediated signaling, β-catenin is a key effector and interacts, as a co-transcription factor, with the DNA binding proteins TCF (T cell factor) and LEF-1 (lymphoid enhancer factor 1) to activate the transcription of Wnt/β-catenin target genes including cyclin D1, c-jun, and c-myc. In this newsletter, the functional regulation of β-catenin and TCF/LEF-1 by PTMs is discussed.

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Nucleation of actin filaments (F-actin) mediated by a complex consisting of the actin nucleation factors Arp2, Arp3, and WASP (VCA domain).

Tu et al. recently characterized the functional relationship between the novel actin bundling protein EFhd2 and actin during lipopolysaccharide (LPS)-induced macrophage migration, an important component of innate immune responses. The authors found that LPS-mediated macrophage migration depends upon EFhd2 and its regulation of actin polymerization and subsequent re-organization of the actin cytoskeleton. EFhd2 and F-actin co-localization in macrophages is essential for LPS’s actions and coincides with a LPS-induced increase in the ratio of F-actin to G-actin in these cells, an effect prevented by knock-out of EFhd2. Under cell-free conditions, EFhd2 increases actin polymerization in a concentration-dependent manner and to a magnitude similar to that induced by the nucleation factors Arp2/3 + VCA domain of the Wiskott-Aldrich syndrome protein (WASP). This suggests that EFhd2’s effects are through a direct regulation of the actin cytoskeleton. Cytoskeleton’s actin polymerization assay kit, G-actin/F-actin in vivo assay kit, Acti-stain 555 phalloidin, Arp2/3 protein complex, and VCA domain of WASP (Cat. # BK003, BK037, PHDH1, RP01P, and VCG03, respectively) were essential reagents in this characterization study. These kits and reagents provided the tools necessary to examine how EFhd2 and its regulation of actin polymerization and cytoskeletal dynamics participate in LPS-induced macrophage motility.

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Tu Y. et al. 2018. EFhd2/swiprosin-1 regulates LPS-induced macrophage recruitment via enhancing actin polymerization and cell migration. Int. Immunopharmacol. 55, 263-271.

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Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin (Cat. # BK003)

G-Actin/F-actin In Vivo Assay Biochem Kit (Cat. # BK037)

Acti-stain 555 phalloidin (Cat. # PHDH1)

Arp2/3 protein complex: porcine brain (Cat. # RP01P)

WASP protein VCA domain: GST tagged: human (Cat. # VCG03)

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The multi-domain, tetrameric p53 protein was discovered in 1979^1-3, first mistakenly described as an oncogene, before its true function as a powerful tumor suppressor was realized^4,5. p53 consists of two N-terminal transactivation domains (TAD1 and TAD2), a proline-rich domain (P-rich), a sequence-specific DNA binding domain (DBD), a linker domain, tetramerization domain (TD), and a lysine-rich, basic C-terminal regulatory (REG) domain⁵ (Fig. 1). As a transcription factor, p53 can regulate the expression of up to 3000 genes  involved in apoptosis, senescence, cell cycle arrest, DNA repair, apoptosis, tumor microenvironment, autophagy, and invasion/metastasis^6-8. p53 functionality is spatiotemporally regulated by up to fifty post-translational modifications (PTMs)that occur within multiple domains^9-12 (Fig. 1). Here, regulation of p53 by ubiquitination, phosphorylation, and acetylation is discussed.

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Protocol video highlighting key steps and tools used in this comprehensive enrichment system.

The use of peer-reviewed, video articles to report technical methodology are an expanding resource that has great benefits compared to text formats, such as providing intricate details that are not effectively captured in written manuscripts. Recently, Horita et al. employed this format to describe a methodology for investigating post-translational modification (PTM) crosstalk.

• Step-by-step instructions to detect acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation in a single lysate.
• Highlights critical components/steps such as a lysate filter system that effectively removes, rather than shears, contaminating genomic DNA. 
• Developed for use with the Signal-Seeker™ PTM detection kits that are effective tools for examining dynamic and endogenous levels of several different PTMs. 

As investigators move forward with PTM research and investigate a specific PTM’s physiologic role, it will be paramount to have effective tools and methodology, like those described in this study, to measure endogenous levels of a PTM for a target protein.

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Horita, H., Law, A., Middleton, K. Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins. J. Vis. Exp. (131), e56912, doi:10.3791/56912 (2018).

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Signal-Seeker™ Phosphotyrosine Detection Kit (30 assay) (Cat. # BK160)

Signal-Seeker™ Ubiquitination Detection Kit (30 assay) (Cat. # BK161)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (30 assay) (Cat. # BK162)

Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay) (Cat. # BK163)

BlastR Rapid Lysate Prep Kit (Cat. # BLR01)

BlastR Rapid Filtration Kit (Cat. # BLR02)

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Intellectual disabilities (e.g., neurodevelopmental disorders, autism spectrum disorders [ASDs]) are associated with abnormal development of dendrites and dendritic spines. ASDs are a complex set of behaviorally defined disorders, characterized by impairments in social interaction, communication, and restricted or stereotyped behaviors. Recent studies estimate that 1% of the world-wide population has an ASD¹. Dendritic spines are comprised of F-actin and the structural and functional plasticity of spines depend upon the dynamic regulation of actin by Rho-family GTPases. Indeed, Rac and PAK effector proteins are essential regulators of normal brain development and function, including dendritic spine initiation, elongation, and branching^2-4. Recent genetic studies revealed that individuals with intellectual disabilities express mutated versions of genes involved in Rho-family GTPase signaling such as a Rho-family GTPase activating protein (GAP), the serine/threonine kinase PAK3, and the Rac/Cdc42 guanine exchange factor (GEF) aPIX5. In addition, the PAK inhibitor FRAX486 is an effective treatment for fragile X syndrome (FXS), the most common inherited form of autism and cognitive disability. FRAX486 reversed dendritic spine and behavioral abnormalities in an in vivo model of FXS⁶. Moreover, Rac1 activation or inhibition of cofilin, an actin depolymerizing protein, rescues ASD-like phenotypes in Shank3 knock-out mice, an in vivo model of ASDs^7-12.

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Figure 1: Schematic representation of the entry points into the MT lumen. Showing, from left to right, a frayed/growing MT plus end capturing TAT and tau molecules, treadmilling, 2 nm2 pores, a 200 nm2 open MT plus end, and a breathing MT lattice.

Balabanian et al. recently investigated the functional regulation of kinesin-1 motility using intact microtubules (MTs) in the form of either a single filament or bundles that were isolated from living COS-7 cells. Post-translational modifications (PTMs), MT bundling, and binding of microtubule-associated proteins (MAPs) are influential modulators of MT stability and motor protein activity. Here, the authors investigated the effects of the acetylation PTM, MT bundling, and binding of the MAP tau on kinesin-1 motility. Cytoskeleton’s SiR-tubulin live cell imaging probe and paclitaxel (Cat. # CY-SC002 and TXD01, respectively) were essential reagents in this kinesin-1 motility study, providing the tools necessary to examine the MT network in living cells and then confirm that it was maintained upon detergent extraction and isolation, followed by the necessary paclitaxel stabilization. The in vitro paclitaxel-stabilized, isolated MT single filaments and bundles faithfully recapitulated important structural attributes of an intact MT network in living cells. This in vitro model enabled the study of how kinesin-1 is functionally regulated by MT architecture, MAP binding, and PTMs such as acetylation. In sum, this model system provides a greater understanding of the physiological regulation of kinesin motors and kinesin-regulated transport of cargoes along MTs.

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Balabanian L. et al. 2017. Acetylated microtubules are preferentially bundled leading to enhanced kinesin-1 motility. Biophys. J. 113, 1551-1560.

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Spirochrome SiR-Tubulin Kit (Cat. # CY-SC002)

Paclitaxel (Taxol) (Cat. # TXD01)

Alpha/beta tubulin heterodimer in complex with tubulin tyrosine ligase, stathmin-4, and the small molecule compound BKM120 (Buparlisib).

Bohnacker et al. recently investigated the primary site of action of the anti-cancer therapeutic BKM120 (a.k.a., Buparlisib), a clinically-advanced phosphoinositide 3-kinase (PI3K) inhibitor. Although PI3K inhibition is considered the primary mechanism of action, some studies report that BKM120 exerts potent off-target effects on tubulin, which raises questions about its substrate. Here, the authors aimed to decipher BKM120’s molecular interactions with tubulin and PI3K to identify its anti-tumorigenic site of action. The authors reported that BKM120’s anti-cancer activity is through mitotic arrest via microtubule destabilization, rather than PI3K inhibition. Chemical derivatives of BK120 were synthesized and examined to separate targeting of PI3K versus tubulin in pursuit of a more potent and selective PI3K inhibitor. The BKM120-derived molecule, PQR309, strongly inhibited PI3K with no detectable effect on tubulin polymerization and/or microtubule stability. Cytoskeleton’s 99% pure porcine brain tubulin, tubulin polymerization assay kit, biotinylated porcine brain tubulin, and TRITC rhodamine-labeled tubulin (Cat.# T240, BK006P, T333P, and TL590M, respectively) were essential reagents in this study, providing the tools necessary to examine how BKM120 and BKM120-derived molecules affected tubulin polymerization and microtubule dynamics as assessed by in vitro assembly and microtubule plus-end tracking assays, with the goal of designing novel, specific, and potent PI3K inhibitors.

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Bohnacker T. et al. 2017. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 8, 14683.

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Tubulin protein (>99% pure): porcine brain (Cat. # T240)

Tubulin polymerization assay using >99% pure tubulin, OD based - Porcine (Cat. # BK006P)

Tubulin protein (biotin): porcine brain (Cat. # T333P)

Tubulin protein (rhodamine): porcine brain (Cat. # TL590M)

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The profilin (Pfn) family of proteins were originally characterized and studied as regulators of actin polymerization. First identified in 1976, these small (14-17 kDa) proteins exist as four isoforms in humans (Pfn1-4). Pfn1 is expressed in most cell types, Pfn2 is primarily localized to the brain, and Pfn3 and Pfn4 are localized to the testes. Pfn expression is essential for the embryonic development of mice. However, forty years later, the role of Pfn proteins in regulating actin polymerization, activating intracellular signal transduction pathways via binding to polyphosphoinositides, regulating microtubule end turnover, binding ligands via poly-L-proline domains, and potentially suppressing tumorigenicity is still being investigated. This newsletter describes the different biological interactions that Pfns are involved in and how these interactions affect actin polymerization.

Profilins and Actin Polymerization

Cellular processes such as trafficking, motility, division, and growth require remodeling of the actin cytoskeleton. Pfns regulate actin polymerization and can both inhibit and promote actin polymerization. Pfns bind to monomeric G-actin in a 1:1 ratio with a binding affinity of 0.1 µM, effectively sequestering the G-actin from incorporation into growing filaments. Notably, the intracellular concentration of Pfns has been estimated to be 10-80 µM, which is not sufficient to maintain the high concentrations of G-actin found in the cell.

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GTPase target characterization of Ras and Rho GEFs using a GEF-mediated exchange assay with BODIPY-FL-GDP to measure changes in fluorescence emission intensity as fluorescent GDP was competed off by unlabeled 500 µM GTP. Rho GEFs Vav1, Vav2, Tiam1, and hDbs were tested against RhoA, Rac1, and Cdc42. GEFs were used at a concentration of 1 µM and GTPases at a concentration of 2 µM.

Rosenberg et al. recently investigated the molecular mechanism responsible for cortactin’s regulation of actin polymerization underlying the maturation and function of invadopodia in breast cancer cells. Release of epidermal growth factor (EGF) stimulates formation of invadopodia, actin-enriched cell protrusions essential for extracellular matrix degradation and cancer cell invasion. The EGF receptor-Src-Arg kinase signaling cascade results in phosphorylation of cortactin. The authors found that two tyrosine-phosphorylated residues on cortactin (Y421 and Y466) bind the SH2 domain of Vav2, a guanine nucleotide exchange factor (GEF) for Rho-family GTPases such as Rac. Tyrosine-phosphorylated cortactin recruits Vav2 to invadopodia, which facilitates their maturation and subsequent invadopodia-mediated matrix degradation and cancer cell invasion. Invadopodial function depends upon re-arrangement of the actin cytoskeleton, all of which requires phospho-cortactin-mediated recruitment of Vav2 to the invadopodia. Furthermore, Vav2’s regulation of actin cytoskeletal dynamics involves Rac3 activation, though the exact role of GTP-bound Rac3 remains unclear. Cytoskeleton’s RhoGEF Exchange Assay Kit and biotinylated actin (Cat. # BK100 and AB07, respectively) were essential reagents in this study, providing the tools necessary to identify Vav2’s substrate targets and effects on actin cytoskeletal dynamics to aid in the development of anti-cancer therapeutics that act by interfering with invadopodia function.

Link to citation:

Rosenberg B.J. et al. 2017. Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells. Mol. Biol. Cell. 28, 1347-1360.

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RhoGEF Exchange Assay (Cat. # BK100)

Actin Protein (Biotin): Skeletal Muscle (Cat. # AB07)

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Acetylation of the epsilon amino group of lysine residues (N^ε-acetylation) is an ancient, highly conserved post-translational modification (PTM) that links acetyl coenzyme A (acetyl-CoA) metabolism and cellular signaling. This occurs largely through the opposing activities of lysine acetyl transferases (KATs) and lysine deacetylases (KDACs). In humans, there are 3 major KAT families (GCN5, CBP/p300, and MYST) that all use acetyl-CoA as an essential cofactor to donate an acetyl group to target lysine residues. There are two KDAC families, the zinc-dependent histone deacetylases (HDAC1-11) and the NAD+-dependent sirtuins (SIRT1-7).

It is well documented that acetylation of nuclear histones plays a major role in regulating chromatin compaction and transcriptional activity wherein acetylation favors a more open, transcriptionally active chromatin. Recent proteomic studies have identified over 4,500 non-histone proteins as targets of acetylation, thereby establishing lysine acetylation as a major global PTM. This PTM is present in many, if not all, cellular compartments, including the nucleus, cytoplasm, cell membrane, and mitochondria. Functionally, reversible lysine acetylation has been shown to regulate enzyme activity, protein-protein interactions, and protein localization and stability. In addition, it plays critical regulatory roles in many cellular processes, including gene expression, cellular metabolism, apoptosis, cytoskeleton regulation, and membrane trafficking.

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Bottom: Rho activation in Swiss 3T3 cells. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1) and nuclear blue DNA staining with Dapi. Cells were activated with Cat.# CN03.

Luo et al. recently discovered the novel GTPase activating protein (GAP), ARHGAP42, through a phosphotyrosine proteomics study. ARHGAP42 de-activates multiple Rho family GTPases, including RhoA. Here, the authors characterized the functional regulation of ARHGAP42 by the non-receptor tyrosine kinase Src and the subsequent downstream effects on ARHGAP42-mediated de-activation of GTP-bound (active) RhoA through engagement of RhoA’s GTPase function. The changes in ARHGAP42 and RhoA activities were correlated with changes in RhoA-regulated actin cytoskeletal dynamics and cell motility in mouse embryonic fibroblasts. ARHGAP42 associates with actin stress fibers and focal adhesions, is auto-inhibited by its N-terminal BAR (Bin/amphiphysin/Rvs) domain, and is activated by Src-mediated phosphorylation of tyrosine residue 376. Following this Src-mediated activation, ARHGAP42 de-activates RhoA through GAP-stimulated GTP hydrolysis which results in increased cell migration mediated by dynamic changes in focal adhesions and the actin cytoskeleton. Cytoskeleton’s RhoA G-LISA activation assay kit and RhoGAP assay kit (Cat. # BK124 and BK105, respectively) were essential reagents in this study, providing the tools necessary to measure the activities of both ARHGAP42 and RhoA to describe the novel functional regulation of ARHGAP42 by the non-receptor tyrosine kinase Src.

Link to citation:

Luo W. et al. 2017. ARHGAP42 is activated by Src-mediated tyrosine phosphorylation to promote cell motility. J. Cell Sci. doi: 10.1242/jcs.197434.

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RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays (Cat. # BK124)

RhoGAP assay (Cat. # BK105)

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The acetylation post-translational modification (PTM) of tubulin occurs primarily on the lysine at position 40 (Lys40) on the alpha-tubulins of assembled microtubules (MTs). The predominant alpha-tubulin acetyltransferase is TAT1/MEC-17. Deacetylation of acetylated tubulin is mediated by histone deacetylase 6 (HDAC6) or SIRT2, the mammalian homolog of silent information regulator 2/sirtuin type 2. Lys40 has long been considered the sole site of acetylation and while other lysine residues on alpha-tubulin and beta-tubulin are targets for acetylation based on proteomic analyses, functional studies focus on the Lys40 residue within the MT lumen. This newsletter discusses tubulin acetylation, MT stability, and the functionality of acetylated MTs (Fig. 1).

Acetylation is a marker for stabilized, long-lived MTs (defined as MTs resistant to nocodazole- and colchicine-induced depolymerization) with a half-life of hours. Notably, acetylation itself does not cause MT stabilization. However, a recent genetic ablation study strongly supports the conclusion that Lys40 acetylation is required for maintaining long-lived, stable MTs in mammalian cells. Loss of TAT1 resulted in a loss of stable, acetylated MTs that are normally present after nocodazole treatment eliminates dynamic MTs. Conversely, TAT1 overexpression significantly increased the amount of nocodazole-resistant (stable) MTs and fibroblasts lacking the tubulin deacetylase HDAC6 have more nocodazole-resistant MTs. The dogma that acetylation is restricted to stable MTs has been revised in recent years as tubulin acetylation is also found on subpopulations of dynamic MTs (half-life of minutes), such as those found in both young and mature neurons.

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Actin is an integral part of the neuronal cytoskeleton as it is involved in the regulation of neuronal polarization, cell morphology, the development of neuronal processes (i.e., growth cones with lamellipodial and filopodial extensions and dendritic spines), intracellular trafficking, and synaptic plasticity (dynamic changes in dendritic spine number and/or morphology)^1-3. Actin’s presence in growth cones and dendritic spines have garnered the attention of scientists for decades; however, actin is also found in neuronal axons, though its presence there has been described as the “black sheep of the neuronal actin family”⁴. This is because the exact details of actin’s structure and role in the axon are unknown. Recently, significant advances have been made in unraveling the structure of axonal actin with the discovery of the periodic membrane skeleton (PMS) by nanoscopic microscopy5 (Fig. 1). This newsletter discusses the discovery, structure, and possible functions of the PMS in axons.

Discovered in 2013, the PMS is a type of cortical actin and the primary component of the actin cortex, a mixture of F-actin and actin binding proteins which supports eukaryotic cells’ plasma membrane and membrane-associated processes such as endo- and exocytosis and cell motility^4,5. In neuronal axons, including the initial segment6, the PMS consists of short actin filaments bundled into evenly spaced rings that wrap around the circumference of the axon with a periodicity of 180-190 nanometers^5-9 (Fig. 1). The short filaments are stabilized by an adducin cap which controls the diameter of actin rings and axons, as well as actin filament growth within the rings^6,10. Adjacent actin rings are secured through cross-linkage by spectrin tetramers (bII in the axon proper and bIV in the axon initial segment)^6,8,11.

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Comparison of BlastR lysis buffer to non-denaturing lysis buffers and detection of total SUMOylation 2/3, acetylation, ubiquitination, and tyrosyl phosphorylation profiles were detected with their respective antibodies.

Recently, Horita et al. examined the post-translational modification (PTM) profile changes in the EGFR→Ras→c-Fos signaling pathway in response to EGF stimulation. PTMs are dynamic and often reversible modifications that alter protein structure and function. While tyrosine phosphorylation (pY) is well-characterized in the EGFR signaling pathway, other PTMs like acetylation (Ac), SUMOylation (SUMO), and ubiquitination (Ub) have not been thoroughly investigated. A novel toolset termed Signal-Seeker™ kits were utilized to investigate the pY, Ac, SUMO, and Ub status of the EGFR signaling axis. All 10 of the previously identified PTMs of the EGFR→Ras→c-Fos signaling pathway were identified, and a novel modification, c-Fos Ac, was also discovered. Importantly, utilizing the toolset enabled investigation of the PTM status of proteins in various cellular compartments that ranged from low to high abundance. The dynamic and endogenous levels of these PTMs were investigated in a single lysis system, providing insight into potential crosstalk between these four PTMs. Cytoskeleton's pY, Ub, SUMO 2/3, and Ac Signal-Seeker™ PTM detection kits (Cat. # BK160, BK161, BK162, and BK163, respectively) were essential reagents in this study, and provide a novel toolset for simple and effective investigation of established and novel PTMs for any target protein.

 

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Actin and Myosin Interactions

Laha et al. recently developed a new thermometric approach for measuring temperature changes of individual proteins on a nanometer (nm) scale. Spatial and temporal resolution of 80 nm and 1 mK, respectively, was achieved by attaching 2 nm cadmium telluride quantum dots (CdTe QDs) directly to bovine cardiac and rabbit skeletal muscle myosins. The goal was to demonstrate that temperature changes of individual motors performing work (i.e., ATP hydrolysis) can be quantified by measuring the corresponding fluorescence intensity shifts of temperature-sensitive CdTe QDs. Heat released by myosin-mediated ATP hydrolysis was quantified as a means of calculating efficiency since heat loss is inversely related to work performed. Using this nanothermometry, rabbit skeletal myosin was more efficient than bovine cardiac myosin at ATP hydrolysis. Nanometer scale sensitivity significantly improved muscle efficiency measurements toward the goal of single cell thermometry to support efforts to provide early diagnosis and treatment of muscle and metabolic diseases on a nanoscale level. Cytoskeleton’s CytoPhos Phosphate Assay, rabbit skeletal muscle myosin, and bovine cardiac muscle myosin (Cat. # BK054, MY02, and MY03, respectively) were essential reagents in this study, providing the tools necessary to measure muscle efficiency as a function of a motor’s temperature change during work.

Laha S.S. et al. 2017. Nanothermometry measure of muscle efficiency. Nano Lett. 17, 1262-1268.

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The ubiquitin E3 ligase Mdm2 (murine double minute 2; human homolog, Hdm2) is well known for its oncogenic activities and as the master regulator of the powerful tumor suppressor p53^1,2. Moreover, Mdm2 may function as an oncogenic protein independent of p53³. Mdm2 is able to inhibit p53-mediated gene expression through two pathways: inhibition of transcriptional activity by direct binding and ubiquitin-mediated degradation via its E3 ligase activity⁴; however, the effectiveness of p53 inhibition by direct Mdm2 binding has been questioned⁵.

Under normal, non-stress conditions, Mdm2 maintains p53 expression and activity at a minimal level to tightly regulate its apoptotic/cell death transcriptional activities. Under conditions of cellular stress, Mdm2-mediated ubiquitination of p53 ceases, allowing p53 to activate transcription of apoptotic genes and those involved in inhibiting cell growth. Much research concludes this is due to Mdm2's auto-inhibition by self-ubiquitination^1-3. However, this story appears to be more complex than originally thought⁵ and involves multiple post-translational modifications (PTMs). Here, we discuss Mdm2's regulation by ubiquitination, SUMOylation, phosphorylation, and acetylation^6-8 (Fig. 1).

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Arf activation by wild-type ARNO GEF in MDCK cells.  ARNO proteins localized with a 9e10 anti-myc primary antibody and CY2-conjugated secondary antibody (green).  F-actin labeled with rhodamine-phalloidin (red).  Cells expressing wild-type ARNO protein have robust lamellipodia (arrows).  Scale bar = 50 microns. Image provided by Dr. Lorraine Santy, Penn State University.

Recently, Rafiq et al. examined Arf1 control of podosome assembly. Podosomes are actin-rich structures surrounded by adhesion and scaffolding proteins that are involved in cell motility and invasion. Podosomes mediate the adhesion of motile cells to the extracellular matrix and are important for the attachment to, and degradation of, the matrix by motile cells. To better understand podosome formation and maintenance, the contribution of Arf1 and its guanine exchange factors (GEFs), as well as the signaling pathways downstream of Arf1 activation, were evaluated in macrophage-like THP1 cells and fibroblasts. Inhibition of Arf1 or the Arf GEF ARNO with small interfering RNAs (Arf1 and ARNO), pharmacological inhibitors (Brefeldin A [BFA] and SecinH3 for Arf1), or expression of dominant-negative mutants (Arf1 and ARNO) significantly impaired podosome formation and maintenance. Conversely, induction of podosome formation increased levels of active Arf1. Arf1 activity was inversely related to RhoA activity as Arf1 inhibition resulted in increased activation of RhoA and myosin IIA filament assembly. Notably, levels of active Rac1 and Cdc42 were unchanged following manipulation of Arf1 activity. Cytoskeleton’s Cdc42 and Arf1 G-LISA activation assay kits (Cat. # BK127 and BK132, respectively) and cell-permeable Rho inhibitor (Cat. # CT04) were essential reagents in this study, providing the tools necessary to measure the activity of multiple GTPases in a quantitative and sensitive manner.

Rafiq N.B.M. et al. 2017. Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO. J. Cell Biol. 216, 181-197.

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Pluripotent stem cells (PSCs), characterized by their unlimited self-renewal and differentiation potential, have garnered special attention as therapeutics because of their capacity to differentiate into any cells of the respective adult organism¹.  There are two general types of PSCs: 1. embryonic stem cells (ESCs) and 2. induced pluripotent stem cells (iPSCs). By definition, PSCs exist in a pluripotent state until differentiation into specialized cells. To maintain stem cells as pluripotent, select transcription factors activate pluripotency-promoting genes and concomitantly suppress differentiation-promoting genes. In turn, the expression level and transactivation ability of these transcription factors are regulated by post-translational modifications (PTMs)^2,3. Three key pluripotent transcription factors are Oct4, Sox2, and Nanog³. Their regulation of transcription is complex with each transcription factor able to function independently of the other while also capable of  auto-inhibition (e.g., Oct4)⁴ or forming a heterodimer whereby one factor is regulated by the complex (e.g., Nanog activity is strictly regulated by the Oct4/Sox2 heterodimer)^5,6. Understanding the precise regulation of the stability (expression level) and transcriptional activity (DNA-binding affinity) of Oct4, Sox2, and Nanog by PTMs is essential in the study of stem cell homeostasis⁷.


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The small GTPase ADP-ribosylation factor 6 (Arf6) belongs to the Arf subfamily of Ras superfamily GTPases. Of the three classes of Arf GTPases, Arf6 is the only member of class III and uniquely localizes to the plasma membrane and endosomes, positioning it to regulate cellular processes dependent upon dynamic changes in the actin cytoskeleton, including endocytosis, exocytosis, trafficking/recycling of membrane-localized proteins, and membrane protrusions (e.g., ruffles). These cellular functions underlie physiological and pathological cell motility and intracellular trafficking. Arf6 cycles between an inactive, GDP-bound state and an active, GTP-bound state to act as a molecular switch in the cellular processes listed above. Activation of Arf6 by exchange of GDP for GTP is mediated by guanine exchange factors (GEFs) while inactivation by GTP hydrolysis is mediated by GTPase activating proteins (GAPs)^1-3. In this newsletter, we discuss the mechanistic roles Arf6 and its GEFs have in cancer cell invasion and metastasis.

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FNR01 image overlay with phase contrast background. Fluorescent fibronectin (Cat. # FNR01) treated MCF10A cells (image kindly provided by A. Varadaraj and M. Karthikenyan, Univ. S.Carolina, Columbia, SC).

Recently, Varadaraj et al. examined transforming growth factor ß (TGF-ß) regulation of the extracellular matrix (ECM) protein fibronectin (FN). Soluble FN dimers polymerize to form insoluble, matrix-associated FN polymers in a process known as fibrillogenesis. The resulting FN fibril network is a scaffold for cell migration, repair, and adhesion mediated by binding to a5ß1 integrin receptors. TGF-ß stimulates ECM remodeling and cell migration through the induction of FN fibrillogenesis, which is necessary for TGF-ß’s effects. Here, the authors found that FN’s role in TGF-ß-mediated ECM remodeling and cell migration can occur via increased FN trafficking, i.e., recycling between the plasma membrane and cytosol. In response to TGF-β, cell surface FN is endocytosed and undergoes Rab11-mediated recycling and subsequent incorporation into fibrils, a process dependent on an interaction between the cytoplasmic domain of the type II TGF-β receptor (TβRII) and a5ß1 integrin. Cytoskeleton’s rhodamine-labeled and biotinylated fibronectin (Cat. # FNR01 and FNR03, respectively) were essential reagents in this interesting study, providing the tools necessary to discover that TGF-ß induces FN trafficking/recycling, a novel process that offers a rapid pathway by which FN can regulate cell migration, wound repair, and fibrosis.

Varadaraj A. et al. 2017. TGF-ß triggers rapid fibrillogenesis via a novel TßRII dependent fibronectin trafficking mechanism. Mol. Biol. Cell. DOI: 10.1091/mbc.E16-08-0601.

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The phosphatidylinositol (3,4,5)-trisphosphate phosphatase and tensin homolog (PTEN) is a tumor supressor protein discovered 20 years ago by two independent laboratories1. PTEN is also known to regulate diverse cellular functions such as adhesion, migration, proliferation, growth, and survival. PTEN is composed of five domains: an N-terminal phosphatidylinositol (4,5)-bisphosphate (PIP2)-binding domain, a catalytic tensin-type phosphatase domain, a C2 tensin-type domain that binds phospholipids, a C-terminal tail domain, and a PDZ-binding domain (Fig. 1). The role of PTEN as a tumor suppressor is attributed to its lipid phosphatase activity which inhibits the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway integral for cell survival and growth by converting phosphatidylinositol (3,4,5)- trisphosphate (PIP3) into PIP2.


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Model: Profile of PD-L1 post-translational modifications and their roles in regulating PD-L1 protein levels.

Recently, Horita et al. profiled four post-translational modifications (PTMs) of the programmed cell death ligand 1 (PD-L1) protein, an important immune checkpoint inhibitor and key target in anti-cancer treatments. Using a set of high-affinity, high-specificity endogenous PTM detection reagents, the authors examined PD-L1's levels of tyrosine phosphorylation, ubiquitination, acetylation, and SUMOylation in A431 cells treated with epidermal growth factor (EGF). These studies led to the novel identification of PD-L1 modified tyrosine phosphorylation, acetylation, and mono- and multi-ubiquitination. Critical temporal studies led to the observation that mono- and multi-ubiquitination preceded an EGF-stimulated increase in total PD-L1 protein expression. Pharmacological inhibition of the EGF receptor (EGFR) activation further demonstrated that mono- and multi-ubiquitination of PD-L1 relies upon EGFR activation.  Importantly, inhibition of ubiquitin E1 activating enzyme blocked any increase in total PD-L1 protein, revealing a potential mechanistic role for ubiquitinated PD-L1 in the regulation of total PD-L1 protein levels. Cytoskeleton's Signal Seeker kits (Cat.# BK160, BK161, BK162), EGF (Cat.# CN02), anti-acetyl lysine antibody (Cat.# AAC01), and anti-tubulin antibody (Cat.# ATN02) were essential reagents in this study, providing the tools necessary for an insightful, novel characterization of PTMs that can regulate PD-L1, an important protein in immune homeostasis, and anti-cancer therapies.

Horita H. et al. 2017. Identifying regulatory posttranslational modifications of PD-L1: A focus on monoubiquitination. Neoplasia. 19, 346-353.

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Worldwide, more than 47 million people have been diagnosed with dementia, and the majority of these cases are caused by Alzheimer’s disease (AD); aside from the social burden, this neurodegenerative disease has an associated cost of 1.09% of the global gross domestic product. Severe cognitive impairment that leads to deficits in skilled movements, language, and recognition are pathophysiological hallmarks of AD. On a molecular level, neuropathological hallmarks include formation of beta-amyloid plaques and neurofibrillary tangles (NFTs) comprised of paired helical filaments of hyper-phosphorylated Tau proteins. This newsletter focuses on the mechanistic control of Tau by post-translational modifications (PTMs) and the development of novel AD therapeutics based on regulating the PTM status of Tau.

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GDP-K-Ras complexed to a phenol moiety (small arrow head; PDB code 4EPT; 2-hydroxyphenyl)(pyrrolidin-1-yl)methanethione) as described in Sun Q. et al. 2012. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew Chem. Int. Ed. Engl. 51, 6140–6143.

Recently, Sakamoto et al. discovered novel peptide inhibitors of the G12D mutant K-Ras GTPase. Of the three Ras isoforms (H-, K-, and N-), K-Ras is considered the most relevant anti-cancer drug target as K-Ras mutations underlie 86% of Ras-linked cancers with 83% of K-Ras mutations at the G12 residue. Development of K-Ras-targeting anti-cancer drugs remains elusive due to the paucity of small, druggable pockets on the GTPase’s surface and picomolar binding affinity between K-Ras and GDP/GTP nucleotides. Here, the authors screened random peptide libraries displayed on T7 phage against recombinant G12D K-Ras in the presence of GDP to identify selective G12D K-Ras inhibitors. Sequence optimization produced a selective G12D K-Ras inhibitor (IC50, 1.6 nM) in a SOS1-mediated GDP/GTP exchange assay. At 30 mM, this inhibitor also reduced proliferation and downstream K-Ras signaling in cancer cells. While less than optimal cell membrane permeability and loss of activity under reducing conditions were shortcomings with this peptide, the discovery of selective G12D K-Ras inhibitors provides a blueprint for the design of future, clinically-relevant K-Ras inhibitors. Cytoskeleton’s SOS1 exchange domain protein (564-1049 amino acids; Cat.# CS-SOS1) was used in the GDP/GTP exchange assay to confirm inhibition of G12D K-Ras activation using BODIPY-FL-GDP.

Sakamoto K. et al. 2017. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology. Biochem. Biophys. Res. Commun. DOI: 10.1016/j.bbrc.2017.01.147.

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Dendritic cells (DCs) are antigen-presenting cells of the mammalian immune system that exist in either an immature, unactivated state or a mature, activated state. Immature DCs (iDCs) patrol peripheral tissues for foreign and/or pathogenic antigens (i.e., antigen sampling), localizing to sites of inflammation. Once there, iDCs find and internalize antigens by phagocytosis, macropinocytosis, or cell surface receptor-mediated endocytosis. During these activities, iDCs fluctuate between fast and slow motility, respectively, presumably to provide an optimal speed for efficient antigen sampling and capture. Concomitant with antigen capture and processing, iDCs undergo maturation, including changes in F-actin and myosin II functional localization which underlies the shift to primarily fast motility. Degraded antigens are presented on the mature dendritic cell surface as major histocompatibility complex (MHC)-II-peptide complexes. Activated DCs migrate chemotactically via lymphatic vessels to naïve T cells in lymphoid organs (e.g., lymph nodes) where the captured antigens are presented to T cells (i.e., immunological synapse), thereby activating them and the adaptive immune system response (Fig. 1).

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Composition and architecture of extracellular matrix (ECM). Basement membrane is a type of ECM composed of laminin and collagen IV fibers embedded within a collagen I-enriched ECM.

FNR01 image overlay with phase contrast background. Fluorescent fibronectin (Cat. # FNR01) treated MCF10A cells (image kindly provided by A. Varadara and M. Karthykenyan, Univ. S.Carolina, Columbia, SC).

Recently, Guzman et al. characterized a novel 3D in vitro model of multicellular cancer cell invasion that offers adjustable physiological and biochemical parameters. Shortcomings of current models are poor microscopic imaging dynamics and inability to study transmigration of cancer cells across the basement membrane (BM) and then invasion of an extracellular matrix (ECM), as happens in vivo. The BM is a type of ECM that surrounds a tumor, formed in a multi-step process initiated by cancer cells binding laminin at the cell surface to form a scaffold upon which type IV collagen polymers form. This new model offers improved imaging dynamics while also recapitulating in vivo tumor cytoarchitecture with an intact, degradable, cell-assembled, sheet-like BM layer embedded in a collagen I-enriched ECM. Cytoskeleton’s HiLyte488-conjugated laminin (Cat. # LMN02) was used to confirm that in vitro BM layer formation paralleled the in vivo process; that is, the laminin scaffold was dispersed across the surface of the multicellular tumor spheroids in a thin, patchy layer, serving as the foundation of the BM layer. This innovative 3D cancer cell invasion model offers researchers the ability to both tease apart molecular mechanisms underlying invasion and test new therapeutics in a physiologically- and biochemically-relevant setting.

Guzman A. et al. 2016. A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane. Biomaterials. 115, 19-29.

Swiss 3T3 cell stained with anti-vinculin (red), DAPI (blue nucleus) and F-actin is stained with Acti-stain™ 488 (green F-actin stress fibers, Cat.# PHDG1).

Recently, Jeganathan et al. examined the role of intersectin-1s (ITSN-1s) in lung cancer proliferation, migration, and metastasis. These cellular processes require actin cytoskeleton re-arrangement typically regulated by RhoA, Rac1, and/or Cdc42 GTPases. ITSN-1s is a multi-domain adaptor protein linking cell surface receptors to intracellular signaling cascades. ITSN-1s’s expression levels are reduced in human lung cancer cells and tissues. Among many findings, the authors report that ITSN-1s down-regulates the epidermal growth factor receptor kinase substrate 8 (Eps8) via ubiquitination and degradation, which in turn decreases the Eps8 and Ras/Rac1 guanine exchange factor mSos1 complex. The Eps8/mSos1 complex activates Rac1 which mediates the subsequent actin cytoskeletal re-arrangements necessary for cancer cell migration and metastasis. Restoration of normal ITSN-1s levels decreases Rac1 activation, increases RhoA activation (leaving Cdc42 activity unaffected), and results in a cytoskeletal network unfavorable to cancer cell migration and metastasis. Cytoskeleton’s RhoA, Rac1, and Cdc42 pull-down activation assays (Cat.# BK036, BK035, and BK034, respectively) were essential for the quantification of RhoA, Rac1, and Cdc42 activities across different levels of ITSN-1s expression. These results suggest that ITSN-1s could serve not only as a novel therapeutic target, but also a prognostic and therapeutic response indicator for lung cancer (and maybe others).

Jeganathan N. et al. 2016. Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis. Mol. Cancer. 15, 59.

Rac activation in Swiss 3T3 fibroblasts. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1). DAPI is the blue nuclear stain. Phalloidin staining shows F-actin-rich lamellipodia. Cells were activated with Cat.# CN04.

Recently, Rom et al. examined molecular pathways involved in leukocyte-mediated neuroinflammation given its causative role in neuronal dysfunction associated with brain injuries and diseases. Neuroinflammation involves a compromised blood-brain barrier (BBB) as leukocytes need to engage brain endothelial cells. To do so, leukocytes utilize integrin adhesion receptors for rolling, arrest, adhesion, and transendothelial migration (TEM), processes requiring Rho-family GTPase-mediated rearrangement of the actin cytoskeleton. Here, the activation of VLA-4 and LFA-1 leukocyte integrins following inhibition of PARP (poly(ADP-ribose) polymerase 1) activity in leukocytes was studied with the goal of preventing BBB breakdown. Using primary human brain microvascular endothelial cells to model the BBB, PARP inhibitors reduced leukocyte adhesion and TEM, concomitant with decreased activation of the two integrins and RhoA and Rac1 GTPases, as well as a reduced F-/G-actin ratio. Cytoskeleton’s RhoA and Rac1 G-LISA activation assays (Cat.# BK124 and BK128, respectively), Acti-stain 488 phalloidin (Cat.# PHDG1), and cell-permeable Rho inhibitor (Cat.# CT04) and Rho/Rac/Cdc42 activator (Cat.# CN04) were essential reagents, allowing for sensitive and reliable quantification of RhoA and Rac1 activation under control and experimental conditions while also measuring dynamic actin cytoskeletal changes. These results suggest novel therapies for protecting BBB integrity following brain disease and injury.

Rom S. et al. 2016. PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier. J. Inflammation. 13, 254.

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Effective treatments for central nervous system (CNS) injuries, diseases, and disorders remain a serious challenge for preclinical research scientists and clinicians This newsletter discusses some compounds that are in clinical trials or proteins/pathways that warrant consideration as therapeutic targets.

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Ras and Rho-family GTPases regulate multiple cellular processes, including development, growth, motility, intracellular trafficking, gene expression, and the cell cycle^1,2. Moreover, dysfunction of these GTPase are correlated with several human diseases (e.g., cancer, neurodegeneration, bacterial pathogenesis)^3-5. Like all GTPases, Ras and Rho GTPases cycle between active (GTP-bound) and inactive (GDP-bound) states. Guanine nucleotide exchange factors (GEFs) regulate GTPase activation, driving the exchange of GDP for GTP in response to a variety of physiological and pathological extracellular signals^1,2. Thus, GEFs are therapeutic targets; however, small molecule inhibitors require hydrophobic pockets for binding which are not typically found on GEFs (or  GTPases). Only recently have novel binding pockets on GEFs and GTPases been discovered^6,7. In this light, the current newsletter explores small molecule inhibitors of Ras (N-, H-, K-Ras) and Rho (RhoA, Rac1, Cdc42) GEFs that inhibit through direct binding.

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Crystal structure of tubulin-stathimine-colchicine complex.

Recently, Zheng et al. identified and characterized a novel tubulin polymerization inhibitor discovered during anti-cancer compound screenings using the epithelial-mesenchymal transition (EMT)-mimetic assay. The lead compound was a nitrobenzoate molecule (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester), designated as compound IMB5046. As part of the in vitro and in vivo characterization process, in vitro tubulin polymerization assays with 97% tubulin/3% microtubule-associated proteins (MAPs) under cell-free conditions demonstrated that the compound inhibits tubulin polymerization, complementing findings from cell culture and mouse xenograft model studies. In addition, surface plasmon resonance (SPR) technology quantified the binding interaction of IMB5046 and tubulin, using biotinylated tubulin and streptavidin-coated sensor chips.  SPR data demonstrated a concentration-dependent, direct interaction and an equilibrium dissociation constant (K_d) of 31.9 µM for IMB5046. Cytoskeleton’s HTS-tubulin polymerization assay kit and >99% pure biotinylated porcine brain tubulin (Cat.# BK004P and T333P, respectively) were essential in this study, providing the necessary research tools to measure the effect of IMB5046 on in vitro tubulin polymerization as well as the binding kinetics with tubulin. In combination with the other findings, a unique, anti-cancer compound with novel chemical structure and the ability to inhibit tubulin polymerization in multidrug-resistant cancer cell lines has been discovered and described.

Zheng Y.-B. et al. 2016. A novel nitrobenzoate microtubule inhibitor that overcomes multidrug resistance exhibits antitumor activity. Sci. Rep. 6, 31472. 

Tubulin Protein (Biotin): Porcine Brain, Cat. # T333P
Tubulin Polymerization Assay Biochem Kit (HTS applications, absorbance): porcine tubulin: 24 assays, Cat. # BK004P

Rho activation in Swiss 3T3 fibroblasts. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1). DAPI is the blue nuclear stain. Phalloidin staining shows F-actin-rich stress fibers. Cells were activated with Cat.# CN04.

Recently, Zhan et al. investigated the molecular pathways underlying human epidermal stem cell (hESC) migration during wound repair to better understand the activity of these cells in the restoration of skin cell and hair follicle homeostasis during wound repair and healing. The authors discovered that nitric oxide (NO) stimulates hESC migration during wound repair and healing, and using the NO donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrated that NO promotes the migration of hESCs in vivo and in vitro. Furthermore, NO-mediated hESC migration in vitro requires cGMP-mediated activation of RhoA and Rac1 (but not Cdc42) signaling pathways. These signaling pathways were examined given that cell migration requires dynamic cell morphology changes which necessitate the re-arrangement of a cell’s actin cytoskeleton, which is strongly regulated by Rho-family GTPases. Cytoskeleton’s Cdc42, Rac1, and RhoA pull-down activation assays (Cat. # BK034, BK035, BK036, respectively) were essential in this study, providing researchers with the necessary research tools to measure Rho-family GTPase activities in a sensitive and consistent manner. These results provide valuable insight into the essential role that Rho-family GTPases have in NO-mediated hESC migration during wound repair and healing to restore proper cellular homeostasis.

Zhan R. et al. 2016. Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signaling. Sci. Rep. 6, 30687.

Cdc42 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays, Cat. # BK034

Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays, Cat. # BK035

RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) - 80 Assays, Cat. # BK036

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The tubulin homolog FtsZ (Filamenting temperature-sensitive mutant Z) protein is an essential prokaryotic cell divison protein. FtsZ is a GTPase that polymerizes in a nucleotide-dependent manner head-to-tail to form single-stranded filaments that assemble into a contractile ring called the Z-ring. This ring forms on the inside of the cytoplasmic membrane where it marks the future site of the septum of a dividing bacterial cell and is dynamically maintained through the course of cell division by continuous and rapid turnover of FtsZ polymers. FtsZ is the first protein to localize at the division site and recruits other proteins involved in bacterial cell division. Besides serving as a scaffold for other cell division proteins, FtsZ itself may exert cytokinetic forces that lead to cell division.

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RhoGAP activity measured as GTP hydrolysis by RhoA protein. Each reaction contained reaction buffer + GTP with the addition of RhoA alone (RhoA), RhoGAP alone (GAP), or RhoA + RhoGAP (RhoA + GAP). Reactions were incubated at 37°C for 20 min. Phosphate generated by hydrolysis of GTP was measured by the addition of CytoPhos™ reagent and reading of absorbance at 650 nm.

Recently, Bendris et al. reported a novel pathway by which the multi-functional, multi-domain scaffold protein sorting nexin 9 (SNX9) controls breast cancer cell invasion and metastasis. SNX9 directly inhibits RhoA activity (and by extension, its downstream effector Rho-associated protein kinase), while exerting no significant direct effect on Cdc42. However, SNX9 directly activates Cdc42's downstream effector, neural Wiskott-Aldrich syndrome protein. The authors evaluated SNX9 for guanine nucleotide exchange factor (GEF) and GTP-activating protein (GAP) activities. SNX9 displayed no direct GEF or GAP activity toward either GTPase. However, SNX9 did inhibit p50GAP-stimulated Cdc42, but not RhoA, GTPase activity. Thus, SNX9 controls the activity of RhoA and Cdc42 in a distinct and opposite manner. These GTPases and/or their downstream effectors control actin cytoskeletal dynamics underlying cell motility, a process integral in cancer cell invasion and metastasis. Cytoskeleton's RhoGAP Assay Kit (Cat. # BK105) was an essential reagent in this study, allowing researchers to determine if SNX9 affects RhoA and Cdc42 GTPase activities by acting either as a GAP itself or influencing the activity of p50GAP. This work advances the understanding of how RhoA and Cdc42 signaling pathways control the cell motility that underlies the invasive and metastatic behavior of cancer cells.

Bendris N. et al. 2016. SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases. Mol. Biol. Cell. 27, 1409-1419. 

RhoGAP assay, 80-160 assays, Cat. # BK105

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The Ras GTPase superfamily, which includes Ras, Rho, Rab, Arf, and Ran subfamilies (among others), has been shown to regulate a wide spectrum of cellular functions. GTPases function as molecular switches, cycling between an inactive GDP-bound form and an active GTP-bound form. The Rho GTPase subfamily includes Rho, Rac, and CDC42, and is believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors...

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Microtubules assembled from HiLyte 488™ labeled tubulin (Cat. # TL488M).

Hoj T.H. et al. 2016. Small molecules revealed in a screen targeting epithelial scattering are inhibitors of microtubule polymerization.J. Biomol. Screen. doi: 10.1177/1087057116651850

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Cardiovascular disease accounts for roughly one in every three deaths in the USA with heart disease accounting for the majority of these cases.  The pathology of heart disease often involves the death or dysfunction of cardiomyocytes, specialized heart cells that produce the contractile, beating function of the heart.  Many different proteins and cell machinery, such as ion channels and pumps, cytoskeletal proteins, and receptors play a significant role in regulating the contractile ability of cardiomyocytes. 

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Kinesin motor proteins regulate mitosis and anterograde cargo transport as exemplified by fast axonal transport (FAT) in neurons. Neurons depend on kinesins for cell cycle regulation, especially the assembly and function of the mitotic spindle, a macromolecular structure composed primarily of microtubules (MTs) that undergo cycles of polymerization and depolymerization to properly segregate duplicate chromosomes into separate daughter cells.

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Rac activation in Swiss 3T3 fibroblasts. F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1). DAPI is the blue nuclear stain. Phalloidin staining shows F-actin-rich lamellipodia. Cells were activated with Cat.# CN04.

Recently, Lao et al. studied how PIAS3, the protein inhibitor of activated signal transducer and activator of transcription 3 (the transcription factor STAT3) regulates the migration, invasion, and activation of fibroblast-like synoviocytes (FLSs), a key component in the pathophysiology of rheumatoid arthritis (RA), specifically joint destruction. Rate of joint destruction is positively correlated with increased FLS motility, invasion, and activity. Here, the authors examined PIAS3-mediated regulation of FLS migration and invasion, and expression of matrix metalloproteinases in RA. Among other findings, PIAS3 knockdown with short hairpin RNA demonstrated that PIAS3 controls lamellipodium formation during FLS migration through activation of Rac1 GTPase as PIAS3 knockdown reduced both the activity of Rac1 and its downstream effector PAK1. Activation of this Rac1 pathway is integral in actin cytoskeleton remodeling which underlies lamellipodium formation and protrusion. Additional results strongly suggest that PIAS3-mediated regulation of Rac1 activation involves SUMOylation (specifically SUMO-1) of Rac1 by PIAS3, as it can act as a SUMO-E3 ligase. Cytoskeleton's Rac1 G-LISA activation assay kit (Cat. # BK128) was an essential reagent in this study, providing a sensitive and quantitative measurement of Rac1 activity following PIAS3 knockdown. These results expand understanding of the molecular pathways regulating FLS migration, invasion, and activation, and the subsequent joint destruction the cells mediate in RA.

Lao M. et al. 2016. Protein inhibitor of activated STAT3 regulates migration, invasion, and activation of fibroblast-like synoviocytes in rheumatoid arthritis. J. Immunol. 196, 596-606.

HeLa CCL-2 cells were grown to 70% confluency at 37°C/5% CO2. Cells were untreated (lanes S1, P1, S2, P2) or treated with 3.3 mM of the tubulin polymerizing drug paclitaxel (i.e., taxol) for 60 min at 37°C/5% CO2 (lanes S3, P3, S4, P4). Cells were lysed and separated into supernatant (S) and pellet (P) fractions and analyzed by western blot quantitation of tubulin protein according to the Microtubules/Tubulin In Vivo Assay Kit (Cat. # BK038) instructions.

Recently, Liu et al. studied how excessive alcohol consumption regulates downstream signaling cascades of mammalian target of rapamycin complex 1 (mTORC1), focusing on the Akt/glycogen synthase kinase-3ß (GSK-3ß)/collapsing response mediator protein-2 (CRMP-2) pathway in rodent nucleus accumbens. Excessive alcohol increases protein levels of the microtubule (MT) binding protein CRMP-2 via mTORC1-mediated translation.  Additionally, Akt is activated, initiating a sequential cascade of GSK-3ß deactivation by Akt-mediated phosphorylation and a subsequent decrease in GSK-3ß -mediated phosphorylation of CRMP-2.  Phosphorylation of CRMP-2 inhibits its binding to MTs and the alcohol-induced reduction in CRMP-2 phosphorylation increases binding between CRMP-2 and MTs, as well as MT asssembly. The authors posit that these alcohol-induced changes in MT binding and protein levels underlie the neuroadaptations (i.e., structural plasticity) that occur in the development and/or maintenance of alcohol-drinking behaviors. Cytoskeleton’s Microtubule Binding Protein Spin-down Assay Kit (Cat. # BK029) and Microtubule/Tubulin In Vivo Assay Kit (Cat. # BK038) were essential reagents in this study, providing quantitation of both MT binding to CRMP-2 and changes in MT content after excessive alcohol consumption. These data suggest that CRMP-2 and its functional relationship with MTs is an essential step in the alcohol-induced neuroadaptations that underlie addiction and addictive behaviors.

Liu F. et al. 2016. mTORC1-dependent translation of collapsing response mediator protein-2 drives neuroadaptations underlying excessive alcohol-drinking behaviors. Mol. Psychiatry. Doi: 10.1038/mp.2016.12.

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Elevations in blood glucose levels are sensed in pancreatic b-cells, which respond through a complex signaling pathway involving mitochondrial-dependent glucose metabolism1. The culmination of this pathway is the mobilization of intracellular insulin-loaded vesicles that fuse with the cell membrane releasing their contents into the...

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Figure 1: Rac1 is activated in serum treated migrating Swiss 3T3 fibroblasts, as shown by staining for F-actin rich lamellipodia with rhodamine phalloidin (Cat. # PHDR1).

Recently, Qin et al. studied the interplay between matrix metalloproteinase-8 (MMP-8) and transforming growth factor beta 1 (TGF-b1) and the effect on each protein’s expression and activity levels in hepatocellular carcinoma (HCC) cells as HCC is responsible for most primary liver cancers.  MMPs are integral for tumor cell metastasis and invasion while TGF-b1 drives cancer progression via epithelial-mesenchymal transition (EMT) and induction of MPP expression.  The authors found that the proteins reciprocally activate each other, leading to increased EMTs and HCC metastasis and invasion in vitro. Moreover, each protein rescues the depleted expression of the other in vitro. In both cases, the PI3K/Akt/Rac1 signaling pathway is the primary mediator as demonstrated pharmacologically. Furthermore, overexpression of MMP-8 or TGF-b1 increases PI3K/Akt/Rac1 pathway activity whereas knockdown exerts the opposite effect.  Cytoskeleton’s Rac1 G-LISA activation assay kit (Cat.# BK128) was an essential reagent in this study as it provided consistent, sensitive, and quantitative measurement of Rac1 activity following manipulation of the expression and activity levels of MMP-8 and TGF-b1. Thus, Rac1 activation mediates the downstream effects of MMP-8/TGF-b1 interplay that results in increased EMT and HCC metastasis and invasion.

Qin G. et al. 2016. Reciprocal activation between MMP-8 and TGF-b1 stimulates EMT and malignant progression of hepatocellular carcinoma. Cancer Lett. 374, 85-95.

A) Human epidermoid carcinoma A431 cells, untreated (top) or treated (bottom) with 5 M TSA (16 h). Acetylated cytoplasmic and nuclear proteins were visualized in green fluorescence.  B) Silver stain of acetyl-lysine immunoprecipitates from wild-type (WT) and muscle-specific knock-out of E1a-binding protein (mKO) mice.

Recently, LaBarge et al. examined the role of the acetyltransferase E1a-binding protein (p300) in skeletal muscle function and metabolism. Reversible acetylation, a well-known post-translational modification, is considered a regulator of mitochondrial metabolism and exercise-induced adaptation in skeletal muscles. This conclusion is based primarily on data derived from studies of deacetylases and skeletal muscle physiology with a dearth of information on how lysine acetyltransferases impact the same muscle physiology. While whole-body heterozygous and homozygous p300 knockout mice have muscle defects (along with neural and cardiac) and die in embryogenesis, to date, the role of p300 in skeletal muscle function has not been studied with a muscle-specific knock-out mouse model. Here, the authors created such an in vivo model and found that knocking out p300 affected neither the development nor function of adult skeletal muscle. Moreover, it was also not required for mitochondrial adaptation induced by endurance exercising. Cytoskeleton’s anti-acetyl lysine antibody (Cat. # AAC01) was an essential reagent in this study as it was used to confirm a functional loss of p300 in the knock-out mice. These mice had a significant reduction in total acetylation levels in skeletal muscle immunoprecipitates from knock-out, compared to wild-type, mice.

LaBarge S.A. et al. 2016. p300 is not required for metabolic adaptation to endurance exercise training. FASEB J. Doi: 10.1096/fj.15-281741.

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The filamentous cytoskeleton is comprised of three distinctive polymer networks: actin filaments, intermediate filaments, and microtubules. Their interplay is responsible for cellular structure, motility, and material transport in order to maintain cellular homeostasis. Also, flexible and rapid cellular responses to external and internal stimuli are possible due...

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F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1) and nuclear blue DNA staining with DAPI. Cells were activated with Cat.# CN04.

Recently, Jones et al. examined the role of Rho-family GTPases in the opposing effects of tetraspanins CD82 and CD37 in bone marrow-derived dendritic cell (BDMC) migration and T cell activation by antigen presentation. Tetraspanins regulate dendritic cell motility and antigen presentation. BMDC activation upregulates CD82, resulting in decreased cell migration and increased T cell activation via stabilization of T cell/dendritic cell interactions. Conversely, CD37 is downregulated and has the opposite response. Deletion of either protein alters cell morphology and the actin and tubulin cytoskeleton. To examine the role of RhoA, Rac1, and Cdc42 in these changes, GTPase activities in cells from either knock-out (CD37 or CD82) or wild-type mice were quantified. Using the cell-permeable Rho/Rac/Cdc42 activator CN04, the authors found that CD82 negatively regulates RhoA while CD37 (but not CD82) regulates Rac1. Both tetraspanins negatively regulate Cdc42. Cytoskeleton's cell permeable Rho/Rac/Cdc42 activator I (Cat.# CN04) and the RhoA (Cat.# BK124), Rac1 (Cat.# BK128), and Cdc42 (Cat.# BK127) G-LISA activation assays were essential reagents in this study. These reagents provided a sensitive measure of Rho-family GTPase activity to discover how these GTPases differentially contribute to the tetraspanin-mediated regulation of BDMC migration and T cell activation in the initiation of adaptive immunity.

Jones E.L. et al. 2016. Dendritic cell migration and antigen presentation are coordinated by the opposing functions of the tetraspanins CD82 and CD37. J. Immunol. Doi: 10.4049/jimmunol.1500357.

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Intermediate filaments (IFs) are one of three filament systems comprising the cytoskeleton of metazoan cells. IFs are highly dynamic structures essential for organizing the actin and tubulin filament systems and regulating cell signaling, motility, structure, and adhesion during interphase and mitosis. The function and localization of IFs are regulated ...

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F-actin is visualized with fluorescent green phalloidin staining (Cat.# PHDG1) and nuclear blue DNA staining with DAPI. Cells were activated with Cat.# CN03.

Recently, Li et al. examined the signaling pathways underlying podoplanin-mediated tumor invasion in oral squamous cell carcinoma (OSCC) tissues and cell lines. Overexpression of podoplanin, a transmembrane glycoprotein, characterizes multiple cancers; however, its exact role(s) in tumor progression/invasion remain unknown. Here, podoplanin overexpression increased tumor cell protrusions (i.e., invadopodia) and F-actin stress fibers in OSCC cells. Concomitantly, RhoA activity decreased whereas Cdc42 activity increased. Correspondingly, podoplanin knockdown reversed these activity patterns. Rac1 activity did not change after any treatments. Additionally, RhoA and Cdc42 engaged in cross-talk (e.g., RhoA inhibition resulted in increased Cdc42 activity). Activated Cdc42 (but not RhoA) was also found to co-precipitate with matrix metalloproteinase-14 (i.e., MT1-MMP) and binding increased with podoplanin overexpression and decreased with knockdown. Cytoskeleton’s Acti-stain 488 phalloidin (Cat.# PHDG1) and RhoA/Rac1/Cdc42 Activation Assay Kit (Cat.# BK030) were essential reagents in this study, providing: 1. detection of morphological changes in the actin cytoskeleton necessary for increased cellular protrusions and stress fibers; and 2. confirmation that this re-organization corresponded with opposing changes in RhoA and Cdc42 activity. Thus, podoplanin-mediated tumor cell protrusion and resulting motility relies upon an activation and inhibition of Cdc42 and RhoA, respectively, which, in turn, mediate the re-organization of the actin cytoskeleton.

Li Y.-Y. et al. 2015. Podoplanin promotes the invasion of oral squamous cell carcinoma in coordination with MT1-MPP and Rho GTPases. Am. J. Cancer Res.5, 514-529.

Temporal regulation of phosphotyrosine-modified Rac1 in response to epidermal growth factor stimulation

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Abstract

The aim of this study was to develop an assay sensitive enough to detect endogenous pY‐modified Rac1 uponstimulation by EGF. The IP assay was used in conjunction with a Rac1 activation assay to follow temporal changesin endogenous Rac1 activation and tyrosyl phosphorylation in response to EGF stimulation of HeLa and A431 cells. Rac1 activation, monitored by PAK‐binding, followed a predicted time course in which...

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Law A., Hong S., Horita H. and Middleton K. 2015. Temporal regulation of phosphotyrosine-modified Rac1 in response to epidermal growth factor stimulation. Mol. Biol. Cell. ASCB Abstract Dec. B1230 / P2126. 

Acti-stain HiLyte 488 phalloidin (Cat.# PHDG1) staining of ruffles characteristic of Rac activation following treatement with a Rho/Rac/Cdc42 activation (Cat.#CN04) in porcine aortic endothelial cells under 40X magnification.

Recently R. Zeineddine et al. examined the molecular signaling pathways underlying cell-to-cell transmission of aggregated copper/zinc superoxide dismutase (SOD1). Misfolding, aggregation, and transmission of SOD1 is implicated in in vitro and in vivo models of Amyotrophic Lateral Sclerosis. Here, the authors examined the signaling pathways involved in macropinocytosis, a form of fluid-phase endocytosis, which provides the means by which macromolecules such as SOD1 aggregates are transmitted cell-to-cell. Macropinocytosis requires membrane ruffling, which in turn, depends upon re-arrangement of the actin cytoskeleton. Thus, the authors examined the activity of a known modulator of the actin cytoskeleton, the Rac1 GTPase, in motoneuron-like (NSC-34) cells. The authors found that exposure of NSC-34 cells to SOD1 aggregates initiates a cascade of sequential events: Rac1 activation > membrane ruffling > macropinocytosis > build-up of SOD1 aggregates intracellularly. Cytoskeleton’s absorbance-based Rac1 G-LISA activation assay kit (Cat. # BK128) was an essential reagent in this study, demonstrating that SOD1 aggregates induce Rac1 activation that leads to the formation of actin-based membrane ruffles. These two events are necessary for propagation of SOD1 protein aggregates (and other disease-associated protein aggregates) via macropinocytosis. These findings suggest therapeutic strategies for treating a multitude of neurodegenerative diseases.

 R. Zeineddine et al. 2015. SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation. Mol. Neurodegener. 10, 57.

Immunofluoresence of HeLa cell in  metaphase with ASM23 Ab (anti-SUMO2/3) and ATN02 Ab (anti-tubulin)

Recently, C.R. Figueiredo et al. examined the mechanism of action underlying the anti-tumor effects of the complementarity-determining region (CDR) C36L1 synthetic peptide derived from the V_L CDR1 of the C36 Fab fragment of the anti-vaccinia immunoglobulin.  This CDR exhibits both anti-tumor activity and functions as a microtubule destabilizing molecule.  Here, the authors investigated whether its microtubule-destabilizing effect is responsible for its anti-tumor activity. The authors found that the peptide induces apoptotic effects on multiple cancer cells both in vitro and in vivo, inhibits tumor cell migration and invasion, and arrests cell cycle primarily in the G2M phase. These effects follow internalization of the C36L1 peptide which then selectively binds to microtubules and destabilizes the tubulin filaments. Microtubule polymerization and depolymerization dynamics were examined in living cells and under cell-free conditions. Cytoskeleton’s fluorescent tubulin polymerization assay kit (Cat. # BK011P) was an essential reagent in this study, complementing the in vivo results and in vitro cell culture data which demonstrated conclusively that the C36L1 peptide targets microtubules in exerting its anti-tumor effects. These findings contribute to the growing and intense field of research focused on developing peptide-based cancer vaccines.

 C.R. Figueiredo et al. 2015. A novel microtubule de-stabilizing complementarity-determining region C36L1 peptide displays anti-tumor activity against melanoma in vitro and in vivo. Sci. Rep. 5, 14310.

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Cytoskeleton, Inc. was founded in 1993 and coincidentally that was the era when the Rho family of small G-proteins was determined to be major regulators of cytoskeletal dynamics. This finding was established in a very direct way by the seminal paper authored by Drs. Anne Ridley and Alan Hall who at that time worked at the Medical Research Council in London(1). Since then, Click to read more

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Microtubules (MTs) are comprised of a/b tubulin heterodimers which have polymerized into cylinderical structures. MTs serve as an essential component of a cell’s cytoskeleton as they regulate and participate in a variety of cellular functions that include motility, morphology, intracellular transport, signal transduction, and cell division (Fig. 1). The cell cycle consists of the sequential G1, S, G2, and M phases with MT polymerization and depolymerization (i.e., MT dynamics) playing a key role in the normal progression of this cycle to insure proper cell division (Fig. 1). The disruption of MT dynamics, and thereby the cell cycle, leads to cell death. As such, MTs are a well-recognized and often-studied target for cancer drug discovery efforts^1-4...

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To maintain homeostasis, cells need to respond to changes in the intracellular and extracellular milieu. Some of the changes have to be acted upon quickly to avoid detrimental effects that can lead to cell damage or even death. One way that cells act is through protein post-translational modifications (PTMs) which enable... Click to read more

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Recent advances in organic chemical synthesis have facilitated the ultimate aim of producing small cell-permeable compounds which can efficiently label the actin cytoskeleton and track its dynamic properties... Click to read more

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Successful execution of mitosis requires exquisite regulation and interplay of a myriad of proteins. Recently, the post-translational modification (PTM) of SUMOylation has emerged as an important functional regulator of mitotic proteins1. SUMO (Small Ubiquitin-like MOdifier) proteins are covalently ligated to ... Click to read more

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The Ras GTPase plays an important role in multiple signal transduction pathways involved in normal cell growth and differentiation as well as several forms of cancer^1,2. The three isoforms of Ras, H-Ras, N-Ras, and K-Ras, were identified over 30 years ago for their oncogenic activation in human tumors^1,2.   Aberrant Ras signaling has been identified in... Click to read more

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YAP1 and the Hippo Pathway

The Hippo signal transduction pathway plays a critical role in the regulation of organ size through the coordinated modulation of cell fate(1). The core pathway memebers include two sets of serine/threonine kinases (MST1/2 and LATS1.2) that act... Click to read more

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RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer^1-4, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion... Click to read more

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The Kinesin Spindle Protein (KSP; a.k.a. Eg5 or KIF11) is a plus end-directed Kinesin-5 (a.k.a. BimC) subfamily member and has been the focus of significant drug development efforts for decades.  Currently, KSP (or its homologs) is a target for anti-mitotics (cancer)^1,2, anti-parasitics (malaria)³, and anti-fungals⁴. As a microtubule (MT) cross-linking enzyme, KSP plays a critical role in mitotic spindle pole separation, and its inhibition results in the formation of monoaster spindles which is thought to lead to mitotic catastrophe and apoptosis (Fig. 1). The targeting of KSP as a treatment for cancer is well-documented^1,2,5,6. The purpose of this newsletter is to briefly discuss KSP homologs as a therapeutic target for parasitic and fungal diseases...

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The activity of Rho family GTPases is regulated temporally and spatially by a variety of direct post-translational modifications (PTMs) that include prenylation, ubiquitination, oxidation, nitrosylation, and phosphorylation (Fig. 1). This newsletter focuses on control of RhoA function through phosphorylation. RhoA is a target for a growing number of kinases and as such, phosphorylation is emerging as a central theme in the regulation of this family of proteins²... Click to read more

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Ras and Rho family GTPases are cytoskeletal small G-proteins that critically regulate multiple actin-dependent cell processes, including development, growth, motility, and intracellular trafficking1,2. Moreover, dysfunction of Ras and Rho family GTPases are correlated with several human diseases (e.g., cancer, neurodegeneration) and these GTPases are... Click to read more

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Protein citrullination (a.k.a. deimination) is a novel arginine-directed post-translational modification (PTM) that results in a permanent change in the targeted protein.  Peptidylarginine deiminases (PADs) mediate the calcium-dependent deimination of the guanidino group of... Click to read more

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Since the discovery of the first kinesin spindle protein (KSP, also known as Eg5, KIF11) inhibitor, monastrol, in 1999 (1), there have been a plethora of articles, 3D structures, and FDA applications based on the same binding site (review [2]). The binding site is called the allosteric monastrol binding site (AMBS) which functionally plays a significant role in transducing chemical energy to movement of the neck-linker region. Drugs that bind this site are non-competitive...

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Recent reports have highlighted a new phase of research for molecular motors. In contrast to five or more years ago, when macromolecular approaches were used¹, recent research has focused on... Click to read more

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Upon translation, proteins are trafficked to their proper subcellular location so that they can perform their physiological functions. One mechanism that mediates correct protein localizationand function is post-translational modifications (PTMs). PTMs include protease cleavage, protein folding, and the attachment of molecules such as... Click to read more

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K-Ras has been identified as the most important Ras protein in cancer research, accounting for over 21% of human cancers. Despite extensive research on these proteins, no effective Ras inhibitor has been identified, earning K-Ras the reputation of an undruggable protein... Here we discuss several leading concepts in the pursuit of identifying viable drug targets to control atypical Ras signaling... Click to read more

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The Kinesin Spindle Protein (KSP; also known as Eg5 or KIF11) is a Kinesin-5 subfamily member and has been the focus of a significant drug development effort throughout the pharmaceutical industry for the last 15 years. KSP plays a critical role in mitotic spindle pole separation, and its inhibition results in the formation of monoaster spindles which is thought to lead to mitotic catastrophe and apoptosis1. From a therapeutic standpoint...

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Redox agents, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are key regulators in a variety of signal transduction pathways, including integrin signaling, extracellular matrix adhesion, and inflammation1-3.  Rho GTPases are also key regulators of many cellular processes, including cell growth, motility, and adhesion4. While redox agents and Rho GTPases operate through a wide array of... Click to read more

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The calcium-sensitive sarcomeric complex is the key mechanochemical transducing unit in muscle cells. It contains myosin, actin, tromomyosin, and three different troponins, one of which, troponin C, binds calcium and facilitates myosin binding to F-actin. The functional sarcomere is controlled by... Click to read more

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Lysine acetylation is a post-translational modification (PTM) crucial for regulating the function and localization of many eukaryotic proteins.  This PTM is reversible, regulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs).  The first evidence of... Click to read more

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Cell migration is an integral part of several biological processes including angiogenesis, wound healing, and immune surveillance(1).  Integrins are αβ heterodimeric transmembrane receptors that link a cell's dynamic interaction with the extracellular matrix (ECM) to the cytoskeletal rearrangements that are necessary to promote cell motility (See figure 1)... Click to read more

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As Cytoskeleton’s Custom Services Department continues to grow and expand its offerings, we wanted to take the opportunity to highlight some past research projects that benefited from work performed by Cytoskeleton’s Custom Services scientists.  The three main foci of Cytoskeleton’s Custom Services are: 1) Compound Screening, 2) Assay Development, and 3) Gene Cloning and Recombinant/Native Protein Purification.  The citations discussed below demonstrate our effectiveness in all three types of custom services...

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Cytoplasmic dynein 1 (hereafter referred to as dynein) is a 1.6 MDa multi-protein complex that serves as the primary ATP-hydrolyzing motor responsible for retrograde axonal transport along microtubules (MTs) in eukaryotic cells (Fig. 1A).   Additionally, dynein is essential for many other cellular processes, including mitochondrial movement, endosomal and lysosomal trafficking, transporting mis-folded proteins bound for degradation, nuclear positioning, and mitosis^1-3 (Fig. 2)... Click to read more

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In eukaryote cells, cytoplasmic dynein complex (CDC) and kinesin-14 family members are the only two types of motors known to move cargo to the minus-end of microtubules (MTs)¹. This function is critical for cell metabolism, structure, and movement. The majority of these processes utilize CDC because of its high fidelity and steady processive movement. CDC’s wide range of roles is impressive and the roles are further defined by ancillary proteins which select cargo or an intracellular location... Click to read more

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