Stabilizes Actin Filaments Research Articles

Abstract Tu146: GJA1-20k Promotes Formation of Mitochondrial Actin Cages

Introduction: GJA1-20k, an N-terminus truncated isoform of the gap junction protein Connexin 43, is stress responsive and mimics ischemia preconditioning, reducing ischemia-reperfusion (IR) injury. GJA1-20k localizes to the outer membrane of mitochondria, stabilizes actin, and mediates an actin mediated mitochondrial protection. The mechanism is not known how GJA1-20k and actin work towards mitochondrial protection. Methods: Cell-free TIRF imaging was performed to study direct interactions between actin and purified GJA1-20k. High-resolution confocal microscopy was applied to visualize GJA1-20k and actin organization around mitochondria in intact HEK cells and cell-free suspensions wherein rhodamine-labeled actin, purified GJA1-20k, and isolated mitochondria were incubated together. Results: Cell-free TIRF imaging establishes that GJA1-20k directly binds to actin , forming both actin clusters and stabilized actin filaments. As expected, live-cell imaging reveals that GJA1-20k is enriched at the mitochondrial outer membrane. The direct binding of GJA1-20k and actin results in a rich collection of actin around mitochondria, as evidenced in both live-cell imaging and cell-free suspensions containing only purified actin, GJA1-20k, and isolated mitochondria. 3D reconstruction reveals that mitochondrial localization of GJA1-20k causes formation of dense actin sheets enveloping mitochondria, which we call “mitochondrial actin cages” which appear to limit the ability of mitochondria to swell under stress conditions. Conclusions: GJA1-20k induced actin cages could be critical to GJA1-20k mediated protection against IR injury. The observed actin around mitochondria could prevent pathological mitochondrial swelling, preserving mitochondrial integrity and function in oxidative stress conditions.

Coactosin-like protein 1 regulates integrity and repair of model intestinal epithelial barriers via actin binding dependent and independent mechanisms.

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation – contributing to cell shape maintenance – is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin – DrbA1-300 – is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Alternative splicing of a single exon causes a major impact on the affinity of Caenorhabditis elegans tropomyosin isoforms for actin filaments.

Tropomyosin is generally known as an actin-binding protein that regulates actomyosin interaction and actin filament stability. In metazoans, multiple tropomyosin isoforms are expressed, and some of them are involved in generating subpopulations of actin cytoskeleton in an isoform-specific manner. However, functions of many tropomyosin isoforms remain unknown. Here, we report identification of a novel alternative exon in the Caenorhabditis elegans tropomyosin gene and characterization of the effects of alternative splicing on the properties of tropomyosin isoforms. Previous studies have reported six tropomyosin isoforms encoded by the C. elegans lev-11 tropomyosin gene. We identified a seventh isoform, LEV-11U, that contained a novel alternative exon, exon 7c (E7c). LEV-11U is a low-molecular-weight tropomyosin isoform that differs from LEV-11T only at the exon 7-encoded region. In silico analyses indicated that the E7c-encoded peptide sequence was unfavorable for coiled-coil formation and distinct from other tropomyosin isoforms in the pattern of electrostatic surface potentials. In vitro, LEV-11U bound poorly to actin filaments, whereas LEV-11T bound to actin filaments in a saturable manner. When these isoforms were transgenically expressed in the C. elegans striated muscle, LEV-11U was present in the diffuse cytoplasm with tendency to form aggregates, whereas LEV-11T co-localized with sarcomeric actin filaments. Worms with a mutation in E7c showed reduced motility and brood size, suggesting that this exon is important for the optimal health. These results indicate that alternative splicing of a single exon can produce biochemically diverged tropomyosin isoforms and suggest that a tropomyosin isoform with poor actin affinity has a novel biological function.

Molecular Basis for Actin Polymerization Kinetics Modulated by Solution Crowding.

Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level.

Actin Depolymerization Factor ADF1 Regulated by MYB30 Plays an Important Role in Plant Thermal Adaptation

Actin filaments are essential for plant adaptation to high temperatures. However, the molecular mechanisms of actin filaments in plant thermal adaptation remain unclear. Here, we found that the expression of Arabidopsis actin depolymerization factor 1 (AtADF1) was repressed by high temperatures. Compared with wild-type seedlings (WT), the mutation of AtADF1 and the overexpression of AtADF1 led to promoted and inhibited plant growth under high temperature conditions, respectively. Further, high temperatures induced the stability of actin filaments in plants. Compared with WT, Atadf1-1 mutant seedlings showed more stability of actin filaments under normal and high temperature conditions, while the AtADF1 overexpression seedlings showed the opposite results. Additionally, AtMYB30 directly bound to the promoter of AtADF1 at a known AtMYB30 binding site, AACAAAC, and promoted the transcription of AtADF1 under high temperature treatments. Genetic analysis further indicated that AtMYB30 regulated AtADF1 under high temperature treatments. Chinese cabbage ADF1 (BrADF1) was highly homologous with AtADF1. The expression of BrADF1 was inhibited by high temperatures. BrADF1 overexpression inhibited plant growth and reduced the percentage of actin cable and the average length of actin filaments in Arabidopsis, which were similar to those of AtADF1 overexpression seedlings. AtADF1 and BrADF1 also affected the expression of some key heat response genes. In conclusion, our results indicate that ADF1 plays an important role in plant thermal adaptation by blocking the high-temperature-induced stability of actin filaments and is directly regulated by MYB30.

How SipA, a toxin from Salmonella, increases stability of F-actin.

Salmonella infection is a disease that is mainly limited to the intestinal tract, but can also be systemic and may be deadly for young children or people with a weakened immune system. Salmonella strains produce protein toxins that target cytoplasmic actin to promote bacterial uptake by a host cell, which is essential for pathogenicity. The SipA protein is a Salmonella effector delivered into the host cell by bacterial type III secretion system. SipA has been reported to stabilize F-actin upon binding, and to promote actin polymerization in vitro. Only very low resolution reconstructions from negative stain existed to show how SipA binds to actin. Here, we present a 3.5Å cryo-EM reconstruction of the SipA425-685/F-actin complex. Our reconstruction shows that SipA binds actin with nearest neighbor exclusion along actin's long-pitch strand. The globular SipA domain binds actin at subunit n through ionic and hydrophobic interactions with both actin strands with nanomolar affinity. The extended C-terminal tail is inserted in between the two actin strands at subunit n+2 preventing the binding of another SipA molecule at this site. Thus, SipA will be bound at n, n+1, n+4, n+5, etc. The C-terminal tail of SipA binds to the same site as two F-actin toxins, phalloidin (Pha) and jasplakinolide (Jasp). All three molecules interact with actin mainly through hydrophobic interactions, however SipA binds F-actin with higher affinity than Pha or Jasp, and outcompetes the latter two in competitive binding assay. The SipA binding involves conformational changes of actin's D-loop which provides additional intra-strand interactions. The observed stabilization of actin filaments upon SipA binding is therefore likely due to the introduction of additional inter- and intra-strand interactions within the actin filament.

Twist response of actin filaments

Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.

The role of Rho GTPase family in cochlear hair cells and hearing.

Rho GTPases are essential regulators of the actin cytoskeleton. They are involved in various physiological and biochemical processes such as the regulation of cytoskeleton dynamics, development, proliferation, survival, and regeneration. During the development of cochlear hair cells, Rho GTPases are activated by various extracellular signals through membrane receptors to further stimulate multiple downstream effectors. Specifically, RhoA, Cdc42, and Rac1, members of the classical subfamily of the Rho GTPase family, regulate the development and maintenance of cilia by inducing the polymerization of actin monomers and stabilizing actin filaments. In addition, they also regulate the normal morphology orientation of ciliary bundles in auditory hair cells, which is an important element of cell polarity regulation. Moreover, the actin-related pathways mediated by RhoA, Cdc42, and Rac1 also play a role in the motility of outer hair cells, indicating that the function of Rho GTPases is crucial in the highly polar auditory sensory system. In this review, we focus on the expression of RhoA, Cdc42, and Rac1 in cochlear hair cells and how these small molecules participate in ciliary bundle morphogenesis and cochlear hair cell movement. We also discuss the progress of current research investigating the use of these small molecules as drug targets for deafness treatment.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Short-Term and Long-Term Sensitization Differentially Alters the Composition of an Anterograde Transport Complex in Aplysia.

Long-term memory formation requires anterograde transport of proteins from the soma of a neuron to its distal synaptic terminals. This allows new synaptic connections to be grown and existing ones remodeled. However, we do not yet know which proteins are transported to synapses in response to activity and temporal regulation. Here, using quantitative mass spectrometry, we have profiled anterograde protein cargos of a learning-regulated molecular motor protein kinesin [Aplysia kinesin heavy chain 1 (ApKHC1)] following short-term sensitization (STS) and long-term sensitization (LTS) in Aplysia californica Our results reveal enrichment of specific proteins associated with ApKHC1 following both STS and LTS, as well as temporal changes within 1 and 3 h of LTS training. A significant number of proteins enriched in the ApKHC1 complex participate in synaptic function, and, while some are ubiquitously enriched across training conditions, a few are enriched in response to specific training. For instance, factors aiding new synapse formation, such as synaptotagmin-1, dynamin-1, and calmodulin, are differentially enriched in anterograde complexes 1 h after LTS but are depleted 3 h after LTS. Proteins including gelsolin-like protein 2 and sec23A/sec24A, which function in actin filament stabilization and vesicle transport, respectively, are enriched in cargos 3 h after LTS. These results establish that the composition of anterograde transport complexes undergo experience-dependent specific changes and illuminate dynamic changes in the communication between soma and synapse during learning.

Septins mediate a microtubule–actin crosstalk that enables actin growth on microtubules

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.

Piperazine Derivative Stabilizes Actin Filaments in Primary Fibroblasts and Binds G-Actin In Silico.

Alzheimer's disease (AD) is characterized by synaptic dysfunction, which is expressed through the loss of dendritic spines and changes in their morphology. Pharmacological compounds that are able to protect spines in the AD brain are suggested to be novel drugs that would be able to slow down the disease progression. We have recently shown that a positive modulator of transient receptor potential cation channel subfamily C member 6 (TRPC6), the compound N-(2-chlorophenyl)-2-(4-phenylpiperazine-1-yl) acetamide (51164), causes the upregulation of postsynaptic neuronal store-operated calcium entry, maintains mushroom spine percentage, and recovers synaptic plasticity in amyloidogenic mouse models of Alzheimer's disease. Here, using confocal microscopy and calcium imaging methods, we present the experimental data indicating that 51164 possesses an alternative mechanism of action. We demonstrated that 51164 can increase the mushroom spine percentage in neurons with the downregulated activity of TRPC6-dependent neuronal store-operated calcium entry. Moreover, we report the binding of 51164 to G-actin in silico. We observed that 51164 interacts with Lys 336, Asp157, and Ser14 of G-actin, amino acids involved in the stabilization/polymerization of the G-actin structure. We showed that interactions of 51164 with G-actin are much stronger in comparison to the well-characterized F-actin stabilizing and polymerizing drug, jasplakinolide. The obtained results suggest an alternative protective mechanism of 51164 that is related to the preservation of actin filaments in vitro.

MNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Comparison of incorporation of wild type and mutated actins into sarcomeres in skeletal muscle cells: A fluorescence recovery after photobleaching study.

The α-actin mutation G15R in the nucleotide-binding pocket of skeletal muscle, causes severe actin myopathy in human skeletal muscles. Expressed in cultured embryonic quail skeletal myotubes, YFP-G15R-α-actin incorporates in sarcomeres in a pattern indistinguishable from wildtype YFP-α-actin. However, patches of YFP-G15R-α-actin form, resembling those in patients. Analyses with FRAP of incorporation of YFP-G15R-α-actin showed major differences between fast-exchanging plus ends of overlapping actin filaments in Z-bands, versus slow exchanging ends of overlapping thin filaments in the middle of sarcomeres. Wildtype skeletal muscle YFP-α-actin shows a faster rate of incorporation at plus ends of F-actin than at their minus ends. Incorporation of YFP-G15R-α-actin molecules is reduced at plus ends, increased at minus ends. The same relationship of wildtype YFP-α-actin incorporation is seen in myofibrils treated with cytochalasin-D: decreased dynamics at plus ends, increased dynamics at minus ends, and F-actin aggregates. Speculation: imbalance of normal polarized assembly of F-actin creates excess monomers that form F-actin aggregates. Two other severe skeletal muscle YFP-α-actin mutations (H40Y and V163L) not in the nucleotide pocket do not affect actin dynamics, and lack F-actin aggregates. These results indicate that normal α-actin plus and minus end dynamics are needed to maintain actin filament stability, and avoid F-actin patches.

CAPG Is Required for Ebola Virus Infection by Controlling Virus Egress from Infected Cells.

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Actin stabilization in cell migration.

Actin is a cytoskeletal filament involved in numerous biological tasks, such as providing cells a shape or generating and transmitting forces. Particularly important for these tasks is the ability of actin to grow and shrink. To study the role of actin in living cells this dynamic needs to be targeted. In the past, such alterations were performed by destabilizing actin. In contrast, we used the natural compound miuraenamide A in living retinal pigmented epithelial (RPE-1) cells to stabilize actin filaments and show that it decreases actin filament dynamics and elongates filament length. Cells treated with miuraenamide A increased their adhesive area and express more focal adhesion sites. These alterations result in a lower migration speed as well as a shift of nuclear position. We therefore postulate that miuraenamide A is a promising new tool to stabilize actin polymerization and study cellular behavior such as migration.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

ABSTRACTInfluenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival.

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments.

Simple SummaryIn this study, we report the effects of depleting cytospin-A (CYTSA), also known as the sperm antigen with calponin homology and coiled-coil domain (SPECC1L) protein, on the proliferation and migration of colorectal cancer (CRC) cells. Mutations in this protein have been previously linked to different developmental disorders. In our studies, depletion of CYTSA in various CRC cells led to significant decreases in proliferation, increases in cell death, and increased formation of multinucleated cells. Knocking down CYTSA also led to severe inhibition of CRC cell migration and invasion. These effects could be related to a significant decrease in the stability of microtubules and alterations in polymerized actin filaments in CYTSA depleted CRC cells. Our studies, for the first time, provide evidence suggesting that targeting CYTSA may be a novel therapeutic strategy for patients with CRC.Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC.