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April 2020 Newsletter: F-Actin Probes in Living Cells

F-Actin Probes in Living Cells

Dynamic remodeling of the actin cytoskeleton [i.e., rapid cycling between filamentous actin (F-actin) and monomer actin (G-actin)] is required for multiple physiological functions, including intracellular transport, cell growth, morphology, motility, trafficking, polarity, cell-to-cell contacts, and cytokinesis1,2. Correspondingly, dysfunctional actin cytoskeletal dynamics are a pathophysiological feature of many human diseases, including those with oncogenic, neurodegenerative, or cardiovascular origins3-9. For these reasons, there is continuing interest in F-actin live cell imaging probes to study actin cytoskeletal dynamics in cell culture models of health and disease (Table 1).


SiR and SPY Actin Probes

The ideal actin visualization tool is a small molecule able to bind F-actin in a sensitive and selective manner, while not disrupting actin re-modeling. In addition, introduction directly into the cell culture medium or tissues without need for transfection or electroporation is advantageous10-13. The new SiR/SPY actin probes fulfill the needs of an “ideal” actin-binding molecule while surmounting most, if not all, of the concerns and shortcomings associated with existing actin probes (Table 1; Figs. 1,2). Initially characterized by Lukinavicius et al.12,13 and introduced commercially in 2014, the SiR and SiR700-actin live cell imaging probes label endogenous F-actin and avoid the need for transfections and over-expression of labeled actin proteins or actin-binding proteins 12,13. SiR/SiR700-actin probes are structurally related to the naturally-occurring F-actin binding molecule jasplakinolide12,13. These F-actin probes utilize the proprietary fluorophore silicon rhodamine (SiR), a bright, photostable far-red dye with little, if any, background signal (Figs. 1,2). Because SiR probes exist in a closed, non-fluorescent state (spirolactone), the probes are self-quenching when unbound to F-actin12,13 (Fig. 3). SiR probes are visualized with standard Cy5 settings (optimal excitation, 650 nm; emission, 670 nm) which confer compatibility with a wide range of genetically-encoded reporter fluorophores (e.g., GFP, m-Cherry)12,13. SPY555-actin is the newest addition to Spirochrome’s family of F-actin live cell imaging probes. SPY555-actin is an improved version of the SiR-actin probes as a lower concentration can be used which offers robust labeling and reduced cytotoxicity and perturbation of actin cytoskeletal dynamics. SPY555-actin is imaged with a standard TMR or Cy3 channel (optimal excitation, 555 nm; emission, 580 nm) using the same staining protocol as for SiR/SiR700-actins. The key features of SiR and SPY actin probes are their cell permeability, fluorogenic character, minimal cytotoxicity, photostability, and compatibility with both standard fluorescence microscopy (e.g., wide-field, confocal) and super-resolution microscopy (e.g., STED, SIM)12-18 (Figs. 1,2). The combination of STED and SiR/SPY-actin probes allows for unparalleled fluorescent visualization of subcellular F-actin structures and their physical characterization in living cells14-18 (Figs. 1,2). SiR-actin probes have been used to examine F-actin in tissue19 and a wide variety of cell types, including (but not limited to) human-induced pluripotent stem cell lines, cardiac cells, endothelial cells, epithelial cells, muscle cells, multiple cancer cell lines, and primary neurons 14,16-18,20-22 (Figs. 1,2).

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Figure 1. MCF10A cells expressing H2B-GFP (blue) in Matrigel (3D culture) stained with SiR-actin (red). Image taken on an inverted LSM microscope. Courtesy of Christian Conrad and Katharina Jechow, Heidelberg.

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Figure 2. SPY505 and SPY555 staining DNA,Actin,Tubulin, and DNA . Photo comes from front page of Spirochrome's website.

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Figure 3. SiR derivatives exist in equilibrium between the fluorescent zwitterionic (open) form (left structure) and the non-fluorescent spiro (closed) form (right structure).

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Fluorescent actin and fluorescent actin-binding domains


The first studies of live cell actin dynamics were performed with fluorescent derivatives of actin protein whch were microinjected into cells50,51. This was a highly effective procedure but the apparatus took a while to setup. Thus overtime, transfections of GFP-actin conjugates became more popular. Fluorescently labeled actin protein or GFP/eGFP-actins worked well with fluorescence recovery after photobleaching (FRAP) microscopy11,23-25.which indicates the dynamic nature of actin cytoskeleton rearrangements. However, GFP/eGFP-actin has several drawbacks10. First, the size of GFP (~28 kDa) can impair polymerization26 and GFP-actin can differentially label F-actin structures10,24. Second, some actin-binding proteins (e.g., formin family nucleators) might sterically hinder incorporation of GFP-actin into actin seeds or growing polymers27,28. Third, there is a relatively high background signal from non-filamentous fluorescent actin29. Finally, expression of eGFP-actin can affect cell behavior30,31.

Another method for actin live cell imaging utilizes yeast- or human-derived actin binding domains fused to GFP, eGFP, or m-Cherry fluorophores10,11,32. The most common genetically-encoded F-actin probes are Lifeact, utrophin (UtrCH), and F-tractin10. Lifeact is a 17 amino acid peptide from yeast Abp14033,34 used for live cell imaging in mammalian and non-mammalian cells 24,34-36. Lifeact has several disadvantages, including the possibility of affecting actin dynamics (so-called Lifeact-induced artifacts) and inhibiting the binding of actin-associated proteins such as cofilin32,37-40. Although Lifeact-GFP binds strongly to F-actin (Kd, 2.2 ± 0.3 μM), its binding affinity for G-actin is 10-fold higher33, resulting in high background fluorescence. Lifeact does not bind all actin-containing structures10,38. Lifeact is introduced into the cell through transfection rather than simply adding it into the medium as is done for the SiR/SiR700/SPY probes. UtrCH is based on the tandem calponin homology domains (CH1 and CH2) of utrophin41 and consists of the first 261 amino acid residues of human utrophin, an actin binding protein42. The CH domains bind to actin with a Kd of ~18 μM43. Utrophin-based probes have been used successfully across a wide range of cell types and species10,32. Similar to Lifeact, at high concentrations, utrophin-based probes can exert deleterious effects on actin cytoskeletal dynamics32,37,44. F-tractin is a 43 amino acid peptide derived from the rat actin-binding inositol 1,4,5-triphosphate 3-kinase A which binds F-actin with a Kd of ~10 μM45,46. Due to its larger size (in comparison to other probes), F-tractin might sterically hinder binding of actin-binding proteins that regulate and/or facilitate polymerization10 and can modify actin-based cellular structures32.

Actin-directed nanobodies and affimer proteins for F-actin

Two new technologies for monitoring actin dynamics in living cells are 1. single-domain antibodies, so-called nanobodies52, and 2. actin “affimers” - synthetic, actin-binding proteins isolated from phage library screens47-49. If not developed correctly, nanobodies can exhibit a high background signal due to G-actin binding10. Recently, three eGFP-fusion actin affimers were described with low micromolar binding affinities for F-actin47, but FRAP microscopy suggests that the eGFP-affimers may preferentially bind to a subset of actin filaments and alter actin organization in the cell47.

Summary

Despite multiple options for visualizing F-actin-based structures in living cells, there is no perfect live cell probe (Table 1). Ideally, the best F-actin probe will be sensitive, selective, fluorogenic, produce a very low background signal, non-toxic, and easily introduced into a wide range of cells across multiple species. It is of paramount importance to confirm that changes in actin cytoskeleton dynamics/structural organization are physiologically relevant and not artifacts of the probe itself. To assist researchers in F-actin live cell imaging studies, Cytoskeleton offers the SiR and SPY actin live cell imaging probes.

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