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• W1977848118 abstract "•Anillin is enriched at the leading edge during neuronal migration and neurite growth•Anillin is essential for Q neuroblast migration and neurite growth•Anillin stabilizes F-actin by antagonizing the F-actin severing activity of Cofilin•Anillin is recruited to the leading edge by active RhoG Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth. Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth. The establishment of the nervous system depends on the accurate positioning of neurons and the correct sprouting and elongation of neurites, which provide the foundation for proper neuronal connectivity and function [1da Silva J.S. Dotti C.G. Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis.Nat. Rev. Neurosci. 2002; 3: 694-704Crossref PubMed Scopus (363) Google Scholar]. Neuronal migration and growth-cone motility are initiated by the activation of membrane receptors through environmental cues and followed by the polarization of the actin cytoskeleton and the extension of the leading edge at the front of the cell [1da Silva J.S. Dotti C.G. Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis.Nat. Rev. Neurosci. 2002; 3: 694-704Crossref PubMed Scopus (363) Google Scholar, 2Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3269) Google Scholar, 3Govek E.E. Hatten M.E. Van Aelst L. The role of Rho GTPase proteins in CNS neuronal migration.Dev. Neurobiol. 2011; 71: 528-553Crossref PubMed Scopus (124) Google Scholar]. Despite the wealth of information about guidance cues, receptors, and the machinery that powers motility, our understanding of the connection between receptor activation and the actin cytoskeleton remains relatively incomplete. Members of the Rho family of small GTPases have emerged as essential regulators of neuronal migration and neurite outgrowth [3Govek E.E. Hatten M.E. Van Aelst L. The role of Rho GTPase proteins in CNS neuronal migration.Dev. Neurobiol. 2011; 71: 528-553Crossref PubMed Scopus (124) Google Scholar, 4Hall A. Lalli G. Rho and Ras GTPases in axon growth, guidance, and branching.Cold Spring Harb. Perspect. Biol. 2010; 2: a001818Crossref Scopus (314) Google Scholar]. These enzymes direct multiple signaling pathways with precise spatial regulation and drive many cellular activities including the reorganization of the actin cytoskeleton: RhoA induces the formation of stress fibers and focal adhesions, Rac promotes actin assembly and the formation of lamellipodia, and Cdc42 is involved in the generation of filopodia [4Hall A. Lalli G. Rho and Ras GTPases in axon growth, guidance, and branching.Cold Spring Harb. Perspect. Biol. 2010; 2: a001818Crossref Scopus (314) Google Scholar, 5Hall A. Rho family GTPases.Biochem. Soc. Trans. 2012; 40: 1378-1382Crossref PubMed Scopus (380) Google Scholar]. In contrast, RhoG (Ras homology growth-related) is one of the less understood Rho family members. Although RhoG was shown to regulate neural development, phagocytosis, and cell growth and survival, the downstream effector of RhoG is largely unexplored [3Govek E.E. Hatten M.E. Van Aelst L. The role of Rho GTPase proteins in CNS neuronal migration.Dev. Neurobiol. 2011; 71: 528-553Crossref PubMed Scopus (124) Google Scholar, 6Alarcón B. Martínez-Martín N. RRas2, RhoG and T-cell phagocytosis.Small GTPases. 2012; 3: 97-101Crossref PubMed Scopus (8) Google Scholar, 7Kwiatkowska A. Didier S. Fortin S. Chuang Y. White T. Berens M.E. Rushing E. Eschbacher J. Tran N.L. Chan A. Symons M. The small GTPase RhoG mediates glioblastoma cell invasion.Mol. Cancer. 2012; 11: 65Crossref PubMed Scopus (55) Google Scholar, 8Franke K. Otto W. Johannes S. Baumgart J. Nitsch R. Schumacher S. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling.EMBO J. 2012; 31: 2908-2921Crossref PubMed Scopus (88) Google Scholar]. Genetic studies revealed that the C. elegans RhoG homolog, MIG-2, is required for neuronal migration and neurite growth, but the underlying cellular mechanisms have not been well addressed [9Zipkin I.D. Kindt R.M. Kenyon C.J. Role of a new Rho family member in cell migration and axon guidance in C. elegans.Cell. 1997; 90: 883-894Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar]. Anillin is an evolutionarily conserved scaffold protein that plays critical roles in the maintenance of appropriate furrow positioning and the formation of a stable midbody during cytokinesis [10Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (567) Google Scholar, 11Hickson G.R. O’Farrell P.H. Anillin: a pivotal organizer of the cytokinetic machinery.Biochem. Soc. Trans. 2008; 36: 439-441Crossref PubMed Scopus (64) Google Scholar, 12Piekny A.J. Maddox A.S. The myriad roles of Anillin during cytokinesis.Semin. Cell Dev. Biol. 2010; 21: 881-891Crossref PubMed Scopus (170) Google Scholar]. Originally isolated as an F-actin binding and bundling protein from Drosophila embryo extracts [13Field C.M. Alberts B.M. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.J. Cell Biol. 1995; 131: 165-178Crossref PubMed Scopus (343) Google Scholar, 14Miller K.G. Field C.M. Alberts B.M. Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.J. Cell Biol. 1989; 109: 2963-2975Crossref PubMed Scopus (114) Google Scholar], Anillin has been implicated in a RhoA-dependent pathway that promotes Anillin’s interaction with numerous furrow components, including actomyosin, septins, microtubules, and the plasma membrane [10Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (567) Google Scholar, 11Hickson G.R. O’Farrell P.H. Anillin: a pivotal organizer of the cytokinetic machinery.Biochem. Soc. Trans. 2008; 36: 439-441Crossref PubMed Scopus (64) Google Scholar, 12Piekny A.J. Maddox A.S. The myriad roles of Anillin during cytokinesis.Semin. Cell Dev. Biol. 2010; 21: 881-891Crossref PubMed Scopus (170) Google Scholar, 15Hickson G.R. O’Farrell P.H. Rho-dependent control of anillin behavior during cytokinesis.J. Cell Biol. 2008; 180: 285-294Crossref PubMed Scopus (99) Google Scholar, 16Maddox A.S. Habermann B. Desai A. Oegema K. Distinct roles for two C. elegans anillins in the gonad and early embryo.Development. 2005; 132: 2837-2848Crossref PubMed Scopus (130) Google Scholar, 17Maddox A.S. Lewellyn L. Desai A. Oegema K. Anillin and the septins promote asymmetric ingression of the cytokinetic furrow.Dev. Cell. 2007; 12: 827-835Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 18Oegema K. Savoian M.S. Mitchison T.J. Field C.M. Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis.J. Cell Biol. 2000; 150: 539-552Crossref PubMed Scopus (236) Google Scholar, 19Piekny A. Werner M. Glotzer M. Cytokinesis: welcome to the Rho zone.Trends Cell Biol. 2005; 15: 651-658Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar]. Although Anillin has been primarily considered to be a central organizer in the cytokinetic machinery, emerging evidence indicates that Anillin has potential roles outside of cytokinesis. In the epithelium of Xenopus embryos, Anillin was demonstrated to regulate the integrity of cell-cell junctions by organizing the junctional enrichment of RhoA-GTP and actomyosin [20Reyes C.C. Jin M. Breznau E.B. Espino R. Delgado-Gonzalo R. Goryachev A.B. Miller A.L. Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin.Curr. Biol. 2014; 24: 1263-1270Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar]. During C. elegans embryogenesis, Anillin appears to be essential for ventral enclosure, a process in which epidermal cells encase the ventral surface of the embryos. However, it is unclear whether Anillin directly regulates epidermal cell migration or whether Anillin acts in a non-autonomous manner to control cytokinesis in the underlying neuroblasts that secrete guidance cues for epidermal cell migration [21Fotopoulos N. Wernike D. Chen Y. Makil N. Marte A. Piekny A. Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.Dev. Biol. 2013; 383: 61-74Crossref PubMed Scopus (21) Google Scholar]. Moreover, human Anillin is strongly expressed in adult neural tissues, suggesting that Anillin may have an unknown function in post-mitotic neurons [22Hall P.A. Todd C.B. Hyland P.L. McDade S.S. Grabsch H. Dattani M. Hillan K.J. Russell S.E. The septin-binding protein anillin is overexpressed in diverse human tumors.Clin. Cancer Res. 2005; 11: 6780-6786Crossref PubMed Scopus (64) Google Scholar]. Here, we show that C. elegans Anillin is asymmetrically localized to the leading edge during neuroblast migration and neurite outgrowth. By generating Anillin conditional knockout animals, we demonstrate that Anillin regulates neuronal migration and neurite growth by antagonizing the F-actin severing activity of Cofilin at the leading edge. We further reveal that Anillin is a novel effector of RhoG/MIG-2 and that active MIG-2 recruits Anillin to the leading edge. Our findings thus reveal a previously unknown pathway in which Anillin links RhoG signaling to the actin cytoskeleton during neural development. C. elegans Q neuroblasts on the left (QL) or the right (QR) of L1 larvae undergo three rounds of asymmetric cell division to generate three distinct neurons and two cells that undergo apoptosis (Figure 1A) [23Ou G. Stuurman N. D’Ambrosio M. Vale R.D. Polarized myosin produces unequal-size daughters during asymmetric cell division.Science. 2010; 330: 677-680Crossref PubMed Scopus (124) Google Scholar, 24Ou G. Vale R.D. Molecular signatures of cell migration in C. elegans Q neuroblasts.J. Cell Biol. 2009; 185: 77-85Crossref PubMed Scopus (36) Google Scholar]. Among the three C. elegans proteins with homology to Anillin, we chose to study the distribution and function of ANI-1 during Q neuroblast development because it has the highest overall conservation with the proteins of other metazoans. Moreover, ANI-1 regulates actomyosin contractility during cytokinesis or polar body emission, and ANI-2 has been implicated to function during oogenesis by stabilizing the intercellular bridge and organizing germline syncytia, whereas ANI-3 appears to be dispensable for development [16Maddox A.S. Habermann B. Desai A. Oegema K. Distinct roles for two C. elegans anillins in the gonad and early embryo.Development. 2005; 132: 2837-2848Crossref PubMed Scopus (130) Google Scholar, 17Maddox A.S. Lewellyn L. Desai A. Oegema K. Anillin and the septins promote asymmetric ingression of the cytokinetic furrow.Dev. Cell. 2007; 12: 827-835Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 21Fotopoulos N. Wernike D. Chen Y. Makil N. Marte A. Piekny A. Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.Dev. Biol. 2013; 383: 61-74Crossref PubMed Scopus (21) Google Scholar, 25Amini R. Goupil E. Labella S. Zetka M. Maddox A.S. Labbé J.C. Chartier N.T. C. elegans Anillin proteins regulate intercellular bridge stability and germline syncytial organization.J. Cell Biol. 2014; 206: 129-143Crossref PubMed Scopus (45) Google Scholar]. We first studied the cellular localization of Anillin during Q cell development. As expected, GFP-tagged Anillin (GFP::ANI-1) accumulates at the cleavage furrow and in the midbody during cytokinesis (Figure S1A; Movie S1), and GFP::ANI-1 then enters the nuclei of the Q, Q.a, Q.p, and Q.pa cells during interphase (Figures 1A and 1B). Quantification of the fluorescence intensity ratio between the cell cortex and the nuclei indicated that GFP::ANI-1 on the cortex is only 0.4- to 0.9-fold of that in the nucleus (Figure 1C), which is consistent with the distribution of Anillin in other cell types during the cell cycle. Unexpectedly, we found that GFP::ANI-1 does not enter the nucleus but rather redistributes to the leading edge of the migrating Q.ap, Q.paa, and Q.pap cells, and that GFP::ANI-1 is 2.7- to 2.9-fold brighter at the cell cortex than in the nucleus (Figures 1A–1C). During the Q.ap dendritic outgrowth, GFP::ANI-1 specifically accumulates in the growth cone (Figure 1D). In both the QL and QR lineages, GFP::ANI-1 shows a similar enrichment at the leading edge or in the growth cone (Figures 1D, 6A, S1C, and S5A; Movies S1 and S2). We did not detect any change in red fluorescence of the mCherry-tagged plasma membrane and histone throughout Q cell development (Figures 1B and 1C). The dynamic changes in GFP::ANI-1 localization suggest that Anillin may function in cell migration and neurite growth. To bypass the essential roles of Anillin in embryonic development and cytokinesis, we used the somatic CRISRP-Cas9 technique to create conditional mutations of ani-1 (Figures 1E and 2) [26Shen Z. Zhang X. Chai Y. Zhu Z. Yi P. Feng G. Li W. Ou G. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development.Dev. Cell. 2014; 30: 625-636Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. We constructed transgenic animals that expressed Cas9 under the control of a heat-shock inducible promoter (Phsp) or Q cell lineage-specific promoters and ubiquitously expressed two single-guide RNAs (sgRNAs) using the U6 gene promoter (PU6). T7 endonuclease I (T7EI)-based assays demonstrated that these transgenic animals generated molecular lesions with the expected sizes at the target loci of ani-1 after heat-shock induction of Cas9 expression (Figure 1F). Heat-shock treatment did not cause defects in embryonic viability or inhibit larval development in wild-type (WT) animals; however, the conditional ani-1 mutant embryos exhibited embryonic lethality with penetrances of 55% (ani-1-sg1) and 35% (ani-1-sg2) and larval arrest phenotypes in 18% of ani-1-sg1 and 35% of ani-1-sg2 animals (Figure 1G), which are consistent with the phenotypes of ani-1 RNAi animals. C. elegans ANI-1 was previously reported to be dispensable for cytokinesis in early embryos, whereas recent studies showed that ANI-1 is essential for cytokinesis in embryonic neuroblasts and vulval precursor cells [16Maddox A.S. Habermann B. Desai A. Oegema K. Distinct roles for two C. elegans anillins in the gonad and early embryo.Development. 2005; 132: 2837-2848Crossref PubMed Scopus (130) Google Scholar, 17Maddox A.S. Lewellyn L. Desai A. Oegema K. Anillin and the septins promote asymmetric ingression of the cytokinetic furrow.Dev. Cell. 2007; 12: 827-835Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 21Fotopoulos N. Wernike D. Chen Y. Makil N. Marte A. Piekny A. Caenorhabditis elegans anillin (ani-1) regulates neuroblast cytokinesis and epidermal morphogenesis during embryonic development.Dev. Biol. 2013; 383: 61-74Crossref PubMed Scopus (21) Google Scholar, 27Bourdages K.G. Lacroix B. Dorn J.F. Descovich C.P. Maddox A.S. Quantitative analysis of cytokinesis in situ during C. elegans postembryonic development.PLoS ONE. 2014; 9: e110689Crossref PubMed Scopus (22) Google Scholar]. We first determined whether ANI-1 regulates Q neuroblast division. We used the gcy-32 promoter to express mCherry in four oxygen sensory neurons, two of which, AQR and PQR (A/PQR), are the progeny of Q.a cells. We could not detect A/PQR in 13%–22% of ani-1 conditional knockouts (Figure 1H), indicating that Q cell asymmetric division or differentiation was defective. To further dissect the role of ANI-1 in Q cell development, we monitored the dynamics of the membrane (Myri::mCherry) and the actin cytoskeleton (GFP::moesinABD) in dividing Q.a and Q.p cells. In WT animals, the Q.a and Q.p cells proceeded through cell division and produced two daughter cells of different sizes; F-actin became enriched at the cytokinetic ring, and contractility was well controlled throughout cytokinesis (Figure 2A for the QR lineage and S2A for the QL lineage; Movie S3). In the ani-1 conditional knockouts, furrow contraction began normally, and F-actin localized to the furrow as it did in WT cells. However, after the furrow had constricted almost to the end, the cleavage furrow regressed, resulting in cytokinesis failure (Figure 2B, 25 min for QR.a and 80 min for QR.p; Figure S2B for QL.a/p; Movie S3). Thus, C. elegans ANI-1 is required for the completion of cytokinesis in Q neuroblasts, which is consistent with the critical roles of Anillin in the cytokinesis of Drosophila and human cell lines [10Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (567) Google Scholar, 12Piekny A.J. Maddox A.S. The myriad roles of Anillin during cytokinesis.Semin. Cell Dev. Biol. 2010; 21: 881-891Crossref PubMed Scopus (170) Google Scholar]. Collectively, we successfully generated C. elegans Anillin conditional knockout animals using somatically expressed CRISPR-Cas9. We studied the function of Anillin in neuronal migration and neurite growth. We showed that 20% of A/PQR neurons reduced their migration distance in ani-1 conditional mutants (Figures 2C and 2E). To determine whether the incomplete penetrance is resulted from worm-to-worm variability, we performed single-worm PCR and T7EI assay in 40 transgenic worms expressing Phsp::Cas9; PU6::ani-1-sg. Among these animals, nine developed Q cell migration phenotypes, and all of them were detected with ani-1 mutations; however, for the rest 31 worms that were normal in cell migration, only six carried ani-1 mutations, indicating that some animals have a strong loss of Anillin function but other do not. We further examined the Q cell migration phenotype in transgenic animals in which Cas9 was expressed under the control of the Q cell lineage-specific promoters Pegl-17 or Pegl-13 (Figure 2E). Pegl-17 is expressed during the early stage of Q cell development [28Mentink R.A. Middelkoop T.C. Rella L. Ji N. Tang C.Y. Betist M.C. van Oudenaarden A. Korswagen H.C. Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans.Dev. Cell. 2014; 31: 188-201Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar], whereas Pegl-13 becomes activated only after the birth of A/PQR neurons [29Feng G. Yi P. Yang Y. Chai Y. Tian D. Zhu Z. Liu J. Zhou F. Cheng Z. Wang X. et al.Developmental stage-dependent transcriptional regulatory pathways control neuroblast lineage progression.Development. 2013; 140: 3838-3847Crossref PubMed Scopus (29) Google Scholar]. We showed that 14% and 10% of A/PQR neurons in Pegl-17::Cas9 and Pegl-13::Cas9 animals, respectively, reduced their migration (Figure 2E). We also found that ∼10% of the ani-1 conditional knockout worms developed neurites whose positioning and extent of growth are perturbed, and the neurites are defective in the formation, guidance, or branching (Figures 2D, 2F, and S2C). In WT animals, AVM and PVM neurons first extend their neurites in the dorsal/ventral (D/V) direction and then elongate along the anterior/posterior (A/P) axis of the animal, and the PQR dendrite sprouts toward the posterior, with the axon sprouting toward the anterior. In ani-1 conditional knockouts, AVM improperly extended its axon to the posterior, the PVM axon skipped the initial D/V growth, and PQR developed its axon toward the posterior (red arrows in Figure 2D). We only examined the phenotypes of neuronal migration and neurite growth in neurons that are mono-nucleated, and these defects may not be secondary due to the failure of cytokinesis. Since neuronal migration and neurite growth occur immediately after cytokinesis, the residual amount of Anillin may be able to support both processes, causing an underestimated penetrance of the phenotypes in mono-nucleated neurons of ani-1 conditional mutants. Next, we examined cell morphology and the actin cytoskeleton during Q cell migration and neurite growth in WT and ani-1 conditional knockouts. In WT animals, QR.ap polarizes toward the anterior and extends a lamellipodium with an enrichment of F-actin at the leading edge, and, after the cell body arrives at the final destination, its leading edge continues to elongate and form the growth cone during neurite growth (Figures 3A and 3C ; Movies S4 and S5). We showed that the F-actin (GFP::moesinABD) was asymmetrically accumulated at the leading edge (Figure 3E). Strikingly, we found that Q cell morphology was altered in ani-1 conditional knockout animals, including the formation of a split leading edge during migration and a branched and misguided growth cone during neurite growth (Figures 3B and 3D; Movies S4 and S5). We further showed that GFP::moesinABD was largely reduced at the leading edge (Figures 3B, 3D, and 3E): the fluorescence intensity ratio between the green F-actin and the red plasma membrane was decreased from 4.2- to 0.3-fold during Q cell migration and from 4.0- to 0.2-fold during neurite growth in ani-1 conditional knockouts (Figures 3F and S3). Thus, Anillin regulates neuronal migration and neurite growth by stabilizing the actin cytoskeleton in the leading edge. We sought to dissect the biochemical pathway through which Anillin establishes appropriate F-actin organization. Previous studies revealed that Anillin acts as a scaffold protein to organize actomyosin [10Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (567) Google Scholar, 11Hickson G.R. O’Farrell P.H. Anillin: a pivotal organizer of the cytokinetic machinery.Biochem. Soc. Trans. 2008; 36: 439-441Crossref PubMed Scopus (64) Google Scholar, 12Piekny A.J. Maddox A.S. The myriad roles of Anillin during cytokinesis.Semin. Cell Dev. Biol. 2010; 21: 881-891Crossref PubMed Scopus (170) Google Scholar]. However, Anillin may function distinctly during cell migration because myosin II does not reside in or function in the physical organization of lamellipodium, even though myosin II plays multiple roles in cell migration [30Vicente-Manzanares M. Ma X. Adelstein R.S. Horwitz A.R. Non-muscle myosin II takes centre stage in cell adhesion and migration.Nat. Rev. Mol. Cell Biol. 2009; 10: 778-790Crossref PubMed Scopus (1313) Google Scholar]. We first examined the distribution of GFP-labeled non-muscle myosin II (NMY-2::GFP) in relation to mCherry-tagged Anillin or F-actin during Q cell migration. We showed that NMY-2::GFP did not co-localize with mCherry-tagged Anillin or F-actin at the leading edge (Figures 4A and 4B ). Instead, NMY-2 was located approximately 0.5 μm behind Anillin or F-actin away from the leading edge of the migrating Q cells, whereas Anillin and F-actin co-localized with each other (Figure 4C). These results are consistent with the established function of myosin II in cell migration and suggest that Anillin may act only with F-actin at the leading edge. To directly characterize the effect of Anillin on the actin cytoskeleton, we prepared the recombinant full-length Anillin protein, its myosin-binding and actin-binding domains (MBD-ABD), or its actin-binding domain (ABD) (Figures 4D, S4A, and S4B). Under a low centrifugation force (see the Supplemental Experimental Procedures), we showed that the presence of Anillin or its MBD-ABD domain increased the amount of preassembled actin filaments in the pellet (Figure 4E; Plastin 3 as the positive control), suggesting that Anillin may organize actin filaments into higher-order structures. This bundling activity was further confirmed by directly visualizing the effects of these Anillin constructs on preassembled actin filaments under a fluorescence microscope (Figure 4F). We next determined whether Anillin stabilizes actin filaments using a dilution-mediated actin depolymerization assay. Without any protection, actin filaments depolymerized into G-actin after dilution, causing a reduction in fluorescence (green lines in Figure 4G). The addition of the full-length Anillin, its MBD-ABD or even its ABD alone inhibited the F-actin disassembly in a dose-dependent manner (Figure 4G). Anillin and its domains did not promote actin assembly in vitro (Figure S4D). Together, our results indicated that the C. elegans full-length Anillin bundles F-actin, as previously demonstrated for Drosophila and vertebrate Anillin [13Field C.M. Alberts B.M. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.J. Cell Biol. 1995; 131: 165-178Crossref PubMed Scopus (343) Google Scholar, 18Oegema K. Savoian M.S. Mitchison T.J. Field C.M. Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis.J. Cell Biol. 2000; 150: 539-552Crossref PubMed Scopus (236) Google Scholar], and that its actin-binding domain alone has a previously uncharacterized role in stabilizing F-actin in vitro. We next examined whether Anillin stabilizes the actin cytoskeleton by counteracting the effect of an F-actin severing factor such as Cofilin [2Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3269) Google Scholar]. The actin-binding domains (ABDs) can play distinct roles in Cofilin-based F-actin severing: although most ABDs can antagonize the severing, the ABD of Fimbrin1 does not and the ABD of Arg (Abl-related protein) even enhances Cofilin-mediated severing [31Skau C.T. Kovar D.R. Fimbrin and tropomyosin competition regulates endocytosis and cytokinesis kinetics in fission yeast.Curr. Biol. 2010; 20: 1415-1422Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 32Courtemanche N. Gifford S.M. Simpson M.A. Pollard T.D. Koleske A.J. Abl2/Abl-related gene stabilizes actin filaments, stimulates actin branching by actin-related protein 2/3 Complex and promotes actin filament severing by cofilin.J. Biol. Chem. 2015; 290: 4038-4046Crossref PubMed Scopus (30) Google Scholar]. We thus determined whether Anillin antagonizes the F-actin severing activity of Cofilin. We first prepared recombinant C. elegans Cofilin/UNC-60 protein (Figure S4C) and showed that the addition of 500 nmol/l (nM) UNC-60 to preassembled F-actin generated breaks along actin filaments (Figure 5A, red arrows; Movie S6). We then added 200 nM full-length Anillin protein, 1.5 μM MBD-ABD, or 2 μM ABD into UNC-60-mediated F-actin severing reactions, and we did not detect the obvious breaks along actin filaments that should have been generated by UNC-60 (Figure 5A; Movie S6). We showed that both UNC-60-mediated filament severing and monomer dissociation were significantly inhibited by Anillin and its MBD-ABD and ABD (Figure 5B). To further explore the antagonism between Anillin and Cofilin in vivo, we examined the migration defects in transgenic animals in which the activity of Anillin or Cofilin was altered. The dephosphorylation of the highly conserved serine 3 in UNC-60 was previously shown to enhance the F-actin severing activity of Cofilin. Although the overexpression of an activated Cofilin/UNC-60(S3A) in WT animals did not cause defects in Q cell migration, UNC-60(S3A) overexpression enhanced Q cell migration phenotypes from 16% to 37% in ani-1 conditional knockouts (Figure 5C). The expression of GFP-tagged Anillin at a low concentration did not affect Q cell migration (Figure 1B and 2 ng/μl plasmid of GFP::ANI-1 for germline injection), however, the overexpression of GFP::ANI-1 with the transformation of 20 and 50 ng/μl plasmid" @default.
• W1977848118 created "2016-06-24" @default.
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• W1977848118 date "2015-05-01" @default.
• W1977848118 modified "2023-10-11" @default.
• W1977848118 title "Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton" @default.
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