The Cytoskeleton Motor Werks™ (CMW) product line is exclusively manufactured and sold by Cytoskeleton. These products facilitate the progress of research and drug discovery in the motor protein area (Funk et al. 2005). We focus on producing highly pure and biologically active kinesin and myosin family proteins of eukaryotic and fungal origin. These reagents are intended for anti-mitotic drug discovery and mechanistic studies of motor activity. The Cytoskeleton Motor Werks™ line also contains several Biochem Kits™, antibodies, and other motor-related reagents (e.g. microtubule stabilizing agent taxol, Cat. # TXD01) and proteins (e.g. pre-formed microtubules, Cat. # MT002).
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Eukaryotic kinesin motor proteins utilize the energy of ATP hydrolysis to move cargoes, such as chromosomes and vesicles, along cytoskeletal microtubule networks . They play a major role in almost all aspects of intracellular transport and are involved in a wide range of physiological processes, including embryonic development , axonal transport , and cell division . The essential role of many kinesins in cell division, and the fact that overexpression of kinesins have been linked to cancers such as retinoblastomas , make them very attractive targets for the development of anti-mitotics. The observation that many kinesins are expressed exclusively during cell division also suggests that they may be superior to current anti-mitotic drug targets such as the ubiquitously expressed tubulins [6,7].
General Structure of Kinesins
The trademark of all kinesins is the presence of a conserved ~340 amino acid motor domain . This domain is divided into two major parts. The first part is a catalytic core region composed of an ATP binding site, which catalyses ATP hydrolysis  and a microtubule (MT) binding site, that tethers the motor to its MT track . The second part is a ~10-40 amino acid neck region which is thought to contribute towards unidirectional motor movement . Beyond the globular motor domain there exists an α-helical coiled-coil region, involved in homo- and hetero-oligomerization of the proteins, which most commonly exist as homodimers. This region is termed the stalk . The stalk often separates the motor domain from a second globular domain termed the tail. The tail domain is thought to be involved in cargo binding.
Kinesin Classification and Structural Diversity
Approximately 150 kinesins have been identified to date in organisms ranging from yeasts (S. cerevisiae contains six kinesins) and fungi to humans (currently 13 kinesins ). Classification is based upon motor domain homologies, particularly in the class specific neck and adjacent regions . It is currently agreed that there are at least nine classes of kinesin [12,13,14]. These are shown in Table 1. The nomenclature (shown in bold in Table 1) is based upon the position of the motor domain within the kinesin protein, hence N-proteins contain a motor domain at the amino-terminal of the protein while C- and M- motors are positioned at the carboxy-terminal and in the middle of the protein respectively .
The recombinant kinesins that are offered by Cytoskeleton have been designed to incorporate the motor domain, including the neck region, plus most of the stalk domain, these constructs are designed to create a normal dimeric structure although this has not been tested by sedimentation assays. The homology of the ~340 amino acid motor region within a given class of kinesin can vary between 74-81% identity in the N-1 class (the most highly conserved, vesicle transport motors) to 46-50% identity in the C class (the most divergent). The percent identity between classes drops to around 35-40% . Structural comparisons of available motor data have revealed that there is significant conformational variability between motors of different kinesin classes and that structural differences are concentrated in six areas, most of which are functionally important in microtubule binding or in linking the core motor to the stalk region [9,11]. Given the relatively high percent variation between the motors of different kinesin classes, it is likely to identify class specific kinesin drugs. This structural evidence is supported by biochemical data which has demonstrated that different classes of Drosophila kinesins exhibit differential utilization of a variety of ATP analogs . This is quite impressive considering that the ATP binding pocket is one of the most highly conserved regions in the motor domain . These experiments show that even within a given species there are clear structural differences between kinesin classes that can be exploited for the purpose of selective drug development. In this regard, it is encouraging that we have been able to detect analog specificity between kinesins in our HTS assay (see Phase I report). We are therefore, confident that we will be able to detect class specific compounds that will be of potential use as anti-cancer drugs.
Representative HTS Target**
Functional Mitotic Inhibition Data
(12 members, 5 phyla)
BimC mutants in Aspergillus inhibited spindle pole body sepatation and caused a lethal phenotype.
In vivo antibody inhibition of HsEg5 by microinjection into HeLa cells caused >80% of cells to arrest in mitosis. These results were confirmed using overexpressed HsEg5 motor mutants.
Involved in cell division probably by binding to chromosomes and helping them align on the metaphase plate. (7 members, 3 phyla)
In vivo antisense and in vitro antibody inhibition and immunodepletion experiments demonstrated the essential role of chromokinesins in spindle organization and chromosome positioning. Motor inhibition led to mitotic block and cell death in Xenopus cells. Deregulation of human Chromokinesin has been linked to retinoblastoma
Involved in chromosome congression on the metaphase plate and possibly chromosome movement at anaphase A
(2 members, 1 phylum)
In vivo overexpression of a motorless CENP-E protein in a human cell line led to the failure of chromosomes to align on the metaphase plate and resulted in a mitotic block . Furthermore, microinjection of CENP-E antibodies into HeLa cells also resulted in mitotic block that lasted between 4 to 17 hrs and resulted in all of the cells entering into apoptosis.
22, 23, 24,25
Involved in anaphase B of mitosis and perhaps cytokinesis.
(5 members, 2 phyla)
A drosophila mutant of the N-VI family demonstrated that this motor is essential for cytokinesis.
Involved in mitotic and meiotic spindle formation, probably by modulating microtubule dynamics. Some members may be exclusively vesicle transporters.
(18 members, 7 phyla)
Mutational analysis in lower eukaryotes (yeast) has demonstrated that null mutants in this class of protein lead to a lethal mitotic block at G2/M.
Required for the onset of anaphase chromosome movement and microtubule dynamics. Some members are exclusively vesicle kinesins. (10 members, 4 phyla)
Inhibition of a mammalian M protein by antisense resulted in disruption of chromosome segregation during anaphase. Overexpression of a motorless mutant construct also resulted in anaphase disruption.
Organelle / vesicle transport
(15 members, 7 phyla)
no role in mitosis
Organelle / vesicle transport, specifically synaptic vesicles and mitochondria
(18 members, 4 phyla )
no role in mitosis
Organelle / vesicle transport specifically anteretrograde vesicle transport.
(13 members, 4 phyla)
no role in mitosis
* The bold classification system is taken from Hirokawa . The classification system in parenthesis is often used in the literature and is taken from Moore and Endow .
** An, Aspergillus nidulans: Hs, Homo sapiens: Dm, Drosophila melanogaster.
Yellow/green shading indicates the classes of kinesins that function exclusively in cell division, light yellow shading indicates classes containing kinesins that function in cell division or mitosis, unshaded area indicates kinesins that are involved in vesicle transport only.
The search for potential anti-fungals requires that we identify structural variations within a class of kinesins, as stated above, the motor region is less variant within any given class of kinesins than between classes. In this regard there are two points of note; first, our major fungal target is the BimC protein which belongs to the most diverse class of kinesins that exhibit approximately 47% identity within the motor region (anything below 60% identity is considered acceptable for differential target specificity), second, we have included the highly variable stalk region in our recombinant protein targets (see Experimental Methods) which offers a relatively protein specific target (<20% identity) . Importantly, antibody inhibition of the stalk region has been shown to inhibit in vivo motor function .
Selection of Kinesin Targets for An HTS Assay
The cellular function of kinesins can be divided into two broad categories; the transport and positioning of membranous vesicles and organelles, and mitotic spindle morphogenesis and chromosome movement . It is not surprising that a given class of kinesins share many functional similarities. It can be seen from Table 1 that four out of nine classes of kinesin motor are exclusively involved in cell division (yellow/green shaded area of Table 1), two classes (C- and M-) contain both vesicle transporters and mitotic motors (light yellow shaded area of Table 1), and three classes (N-I, NIII and N-IV) function exclusively in vesicle transport (unshaded area of Table 1).
We have selected a human homolog from each kinesin class [6,17,21,22,26,28,29,30,39] and two fungal kinesins from Aspergillus, a human pathogen , as the basis for the HTS assay (these are shown in Table 1). By screening each protein with a compound library it is possible to generate a primary database that will allow identification of compounds that selectively target the cell division specific class of human kinesin (darkly shaded area of Table 1) while having no effect upon vesicle transport kinesins. Such compounds are most likely to be effective anti-mitotics in the treatment of human diseases such as cancer. Likewise, compounds reacting specifically with the Aspergillus kinesins can be pursued as potential anti-fungals.
Kinesins As Valid Anti-Mitotic Targets
Before beginning an expensive drug discovery program, one has to critically assess the suitability of the proposed protein/s to meet the criteria of a valid drug target. Evidence that kinesins will make excellent targets for anti-mitotic drug development has come from a range of experimental approaches, including mutational analysis , antibody inhibition experiments [7,32] and antisense inhibition of kinesin activity . This body of evidence is summarized in Table 1 where it can be seen that functional inhibition of a given mitosis specific kinesin results in inhibition of cell division. For example, in vivo inhibition of HsEg5 by microinjection of anti-Eg5 antibodies into HeLa cells caused >80% of cells to arrest in mitosis. These results were confirmed using overexpressed HsEg5 motor domain mutants . In another set of experiments, in vivo overexpression of a motorless CENP-E protein in a human cell line led to the failure of chromosomes to align on the metaphase plate and resulted in a mitotic block . Furthermore, microinjection of CENP-E antibodies into HeLa cells also resulted in mitotic block that lasted between 4 to 17 hours and resulted in all of the cells entering into apoptosis . As both of these proteins are included in our HTS assay, we are very hopeful that compounds directed against these motors will be therapeutically relevant. Interestingly, class N-V kinesin (Table 1) contains the human chromokinesin protein which has been linked to retinoblastoma . The fact that antisense and antibody inhibition of the Xenopus homolog of this motor lead to a mitotic block and cell death  is an encouraging indication that drugs targeted to the human chromokinesin may be therapeutically useful in the treatment/prevention of retinoblastoma.
In lower eukaryotes, mutational analysis has served to elucidate the role of mitotic kinesins. Often mutations in a single kinesin will lead to a mitotic block and a lethal phenotype. This is the case for the AnBimC protein that we have selected as an anti-fungal target . However, in certain cases, functional redundancy has been demonstrated in the form of two highly homologous genes carrying out the same function . In these instances, both genes must be knocked out in order to produce the mitotic phenotype. Of the mitosis specific proteins that we have selected for our representative kinesin panel, those that have been inhibited in vivo (three out of five, see Table 1) exhibit a definite phenotype indicating that they do not exhibit functional redundancy, or that their related homolog is also inhibited. The remaining two, HsChromokinesin and HsMKLP1 may exhibit functional redundancy in vivo and this possibility should be taken into account if cell based secondary assays do not result in a mitotic arrest phenotype.
Importantly, all available data suggests that antibodies directed against mitotic kinesins specifically affect the mitotic process but have no effect upon kinesin vesicle transport functions . Indeed, inhibition of a specific kinesin often induces a very cell cycle specific phenotype. For example, inhibition of the motor region in the M-class kinesin MCAK resulted in no effect upon spindle assembly but did inhibit chromosome movement . This data suggests that specific inhibitors of kinesin motors will inhibit the mitotic process very specifically without affecting other critical cellular functions.
Possible Advantages of Kinesin Targets Over Current Anti-Mitotic Targets
The microtubule spindle protein tubulin is the major target of current anti-mitotics such as taxol and vinblastine [43,44]. It is generally accepted that these drugs work by directly suppressing microtubule dynamics during mitosis [45,46]. Specificity towards dividing cells is favored due to the fact that microtubule dynamics are much greater in mitotic cells than quiescent ones. Drug treatment results in a mitotic block during which time the cells enter into the apoptotic pathway and die . Due to the nature of their mechanism of action, all anti-mitotics, including taxol and vinblastine, will, to some extent, adversely affect normal mitotic cells such as those present in the thymus, testis, small intestine, colon and placenta . However, anti-tubulins also suffer from dose limiting side-effects of neurotoxicity due to the fact that tubulin is present in high amounts in neuronal tissue [49,50].
A major advantage of the mitotic kinesins as potential anti-mitotic drug targets is the fact that their expression is often tightly regulated to coincide with the mitotic event. For example, HsMCAK is expressed only in proliferating cells where expression has been shown to be tightly regulated at transcriptional level ; HsEg5 only associates with microtubules during mitosis it is not associated in vivo with MTs during interphase ; chromokinesins are expressed exclusively in proliferating cells where they are short lived proteins, probably regulated by a cyclin like degradation mechanism ; CENP-E has been demonstrated to bind to kinetochores immediately after nuclear breakdown and remain fully bound until anaphase B, when it relocalizes to the anaphase B spindle and is subsequently degraded via a cyclin-like pathway . The tight regulation of mitotic kinesin expression predicts that these will be highly specific anti-mitotic targets with minimal dose limiting side effects.
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7) Endow, S., Chandra, R., Komma, D., Yamamoto, A., Salmon, E. Mutations of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis. J. Cell Sci. 107:859-867 
8) Meluh, P., Rose, M. KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60:1029-1041 
9) O’Connell, M., Meluh, P., Rose, M., Morris, R. Suppression of the bimC4 mitotic spindle defect by deletion of klpA, a gene encoding a KAR3-related kinesin-like protein in Aspergillus nidulans. J. Cell Biol. 120:153-162 
10) Hoang, E., Whitehead, J., Dose, A., Burnside, B. Cloning of a novel C-terminal kinesin (KIFC3) that maps to human chromosome 16q13-q21 and thus is a candidate gene for Bardet-Biedl syndrome. Genomics 52:219-222 
11) Kim, I., Jun, D., Sohn, U., Kim, Y. Cloning and expression of human mitotic centromere-associated kinesin gene. Biochem. Biophys. Acta. 1359:181-186 
12) Maney, T., Hunter, A., Wagenbach, M., Wordeman, L. Mitotic centromere associated kinesin is important for anaphase chromosome segragation. J. Cell Biol. 142:787-801 
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14) Enos, A., Morris, N. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans. Cell 60:1019-1027 
15) Blangy, A., Chaussepied, P., Nigg, E. Rigor type mutation in the kinesin related protein HsEg5 changes its subcellular localization and induces microtubule bundling.
16) Sawin, K., LeGuellec, K., Phillippe, M., Mitchinson, T. Mitotic spindle organization by a plus end directed microtubule motor. Nature 359:540-543 
17) Blangy, A., Lane, H., d’Herin, P., Harper, M., Kress M., Nigg, E. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin related motor essential for bipolar spindle formation in vivo. Cell 83:1159-1169 
18) Walczak, C., Vernos, I., Mitchison, T., Karsenti, E. A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity. Curr. Biol. 8: 903-913 
19) Vernos, I., Raats, J., Hirano, T., Heasman, J., Karsenti, E., Wylie, C. Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. Cell 81:117-127 
20) Tokai, N., Fujimoto, A., Toyoshima, Y., Tsukita, S., Inoue, J., Yamamota, T. Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle. EMBO J. 15:457-567 
21) Yan, R.T., Wang, S.Z. Increased chromokinesin immunoreactivity in retinoblastoma cells. Gene 189:263-267 
22) Schaar, B., Chan, G., Maddox, P., Salmon, E., Yen, T. CENP-E function at kinetochore is essential for chromosome alignment. J. Cell Biol. 139:1373-1382 
23) Yen, T., Li, G., Schaar, B., Cleveland, D. CENP-E is a putitive kinetochore motor that accumulates just before mitosis. Nature 359:536-539 
24) Wood, K., Sakowicz, R., Goldstein, L., Cleveland, D. CENP-E is a plus end directed kinetochore motor required for metaphase chromosome alignment. Cell 91:357-366 
25) Lombillo, V., Nislow, C., Yen, T., Gelfand, V., McIntosh, R. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro. J. Cell Biol. 128:107-115 
26) Nislow, C., Lombillo, V.A., Kuriyamu, R., McIntosh, R. A plus end directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature 359:543-547 
27) Adams, R.R., Tavares, A.A., Salzberg, A., Bellen, H., Glover, D. Pavoretti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes & Dev. 12:1483-1494 .
28) Wright, B., Terasaki, M., Scholey, J. Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection. J. Cell Biol. 123:681-689 
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31) Enos, AP & Morris, NR. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in Aspergillus nidulans. Cell 60: 1019-1027 .
32) Yen, TJ., Li, G., Schaar, BT., Szilak, I. and Cleveland, DW. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature 359(6395): 536-539 .
33) Wang, SZ & Alder, R. Chromokinesin: a DNA-binding, kinesin-like nuclear protein. J.Cell Biol. 128:761-768 .
34) Blangy, A., Lane, HA., d’Herin, P., Harper, M., Kress, M. and Nigg, EA. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83(7): 1159 .
35) Navonne, F., Niclas, J., Hom-Booher, N., Sparks, L., Bernstein, H., McCaffrey, G and Vale, R. Cloning and expression of a human kinesin heavy chain gene: Interaction of the C-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J. Cell Biol. .
36) Telford, EA., Wightman, P., Leek, J., Markham, AF., Lench, NJ. and Bonthron, DT. cDNA cloning, genomic organization, and chromosomal localization of a novel human gene that encodes a kinesin-related protein highly similar to mouse Kif3C. Biochem. Biophys. Res. Comm. 242(2): 407 .
37) Hoang, E., Whitehead, J., Dose, A., Burnside, B. Cloning of a novel C-terminal kinesin (KIFC3) that maps to human chormosome 16q13-q21 and thus is a canidate gene for Bardet-Biedl syndrome. Genomics 52: 219 .
38) Kim, IG., Jun, DY., Sohn, U. and Kim, YH. Cloning and expression of human mitotic centromere-associated kinesin gene. Biochim. Biophys. Acta 1359(3): 181 .
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44) Nislow, C., Lombillo, V.A., Kuriyama, R. and McIntosh, J.R. (1992). A plus-end directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature 359:543-547.
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47) Wood, K., Sakowicz, R., Goldstein, L., Cleveland, D. CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell 91:357-366 .
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Cytoskeleton's motor protein products have been cited hundreds of times over the past two decades. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.
Borysov et al., 2011. Alzheimer A-beta disrupts the mitotic spindle and directly inhibits mitotic microtubule motors. Cell Cycle. 10, 1-14.
Borysov et al., 2011. Alzheimer A-beta disrupts the mitotic spindle and directly inhibits mitotic microtubule motors. Cell Cycle. 10, 1-14.
Ezratty, E. J., Partridge, M. A. and Gundersen, G. G. (2005). Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat. Cell Biol. 7, 581-590.
Gomes, E. R., Jani, S. and Gundersen, G. G. (2005). Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121, 451-463.
Kosturko, L. D., Maggipinto, M. J., D'Sa, C., Carson, J. H. and Barbarese, E. (2005). The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2. Mol. Biol. Cell 16, 1938-1947.
Teckchandani, A. M., Birukova, A. A., Tar, K., Verin, A. D. and Tsygankov, A. Y. (2005). The multidomain protooncogenic protein c-Cbl binds to tubulin and stabilizes microtubules. Exp. Cell Res. 306, 114-127.
Answer 1: Kinesin motor proteins use microtubules (MTs) as a substrate to orchestrate a wide range of kinetic events within a cell. They have been shown to move cargoes such as chromosomes and vessicles along MT tracks. Kinesins operate by utilizing the energy of ATP by hydrolysis, an activity that is greatly enhanced in the presence of MTs. To screen drugs that modulate the interactions between motor proteins and MTs, a MT-activated kinesin ATPase assay can be used as a test for the MT-activated ATPase activity of kinesins. Cytoskeleton developed two such assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) levels generated by microtubule-activated kinesin adenosine triphosphatase (ATPase) activity. These two kinesin ATPase biochem assay kits (Cat. # BK053 and BK060) provide MTs and kinesin heavy chain (KHC) protein along with the necessary buffers and reagents to measure Pi production as a means of screening drugs that modulate kinesin and/or MT functional interactions. These kits are useful for discovering kinesin inhibitors and activators (Cat. # BK053 and BK060) as well as determining Vmax and Kcat values for a kinesin motor protein (Cat. # BK060).
Answer 2: Motor proteins utilize microtubules as a substrate, meaning that motor proteins are one type of microtubule-associated protein (MAP). To confirm that the putative motor protein binds with microtubules, Cytoskeleton’s microtubule binding protein spin down assay kit (Cat. # BK029) is available. The protein to be evaluated will need to be visualized by either Coomassie Blue or silver staining or with an antibody to either the protein itself or a tag conjugated to the protein (his, myc, etc.). A non-hydrolyzable ATP analog, e.g. AMPPNP, is usually used at 5 or 10 mM to maintain microtubule attachment. Microtubules also activate the ATPase activity of motor proteins which can be measured with one of our kinesin ATPase assays (Cat. # BK053 or BK060). These kits are useful for discovering kinesin inhibitors and activators (Cat. # BK053 and BK060) as well as determining Vmax and Kcat values for a kinesin motor protein (Cat. # BK060). Cytoskeleton, Inc. also offers a Biochem Kit (Cat. # BK027) which allows the visualization of motor protein motility using fluorescently-labeled microtubules.