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• W2086681436 abstract "Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement. Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement. Microtubules (MTs) and filamentous actin (F-actin) control diverse cellular functions, including cell shape, cell division, intracellular transport, adhesion, and movement. Increasing evidence suggests that intricate molecular interactions exist between these two seemingly distinct cytoskeletal networks (Goode et al., 2000Goode B.L. Drubin D.G. Barnes G. Functional cooperation between the microtubule and actin cytoskeletons.Curr. Opin. Cell Biol. 2000; 12: 63-71Crossref PubMed Scopus (406) Google Scholar, Palazzo and Gundersen, 2002Palazzo A.F. Gundersen G.G. Microtubule-actin cross-talk at focal adhesions.Sci. STKE. 2002; 2002: PE31PubMed Google Scholar, Rodriguez et al., 2003Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Conserved microtubule-actin interactions in cell movement and morphogenesis.Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (681) Google Scholar, Yarm et al., 2001Yarm F. Sagot I. Pellman D. The social life of actin and microtubules: Interaction versus cooperation.Curr. Opin. Microbiol. 2001; 4: 696-702Crossref PubMed Scopus (33) Google Scholar). The coordination of cytoskeletal dynamics is particularly important for cell migration and adhesion, processes that are intrinsic and essential features of tissue morphogenesis and physiology. The complex, multistep process of directed cell migration requires integrated activities of cytoskeleton, membrane, and cell/extracellular matrix (ECM) adhesions (Lauffenburger and Horwitz, 1996Lauffenburger D.A. Horwitz A.F. Cell migration: A physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3142) Google Scholar). Recent studies have pointed to an importance of MTs and coordinated MT/actin dynamics in cell migration (Palazzo and Gundersen, 2002Palazzo A.F. Gundersen G.G. Microtubule-actin cross-talk at focal adhesions.Sci. STKE. 2002; 2002: PE31PubMed Google Scholar, Rodriguez et al., 2003Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Conserved microtubule-actin interactions in cell movement and morphogenesis.Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (681) Google Scholar). It has long been known that MTs can be transported rearward in the lamellae of migrating cells, and this movement depends on actomyosin activity (Mikhailov and Gundersen, 1995Mikhailov A.V. Gundersen G.G. Centripetal transport of microtubules in motile cells.Cell Motil. Cytoskeleton. 1995; 32: 173-186Crossref PubMed Scopus (58) Google Scholar, Waterman-Storer and Salmon, 1997Waterman-Storer C.M. Salmon E.D. Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.J. Cell Biol. 1997; 139: 417-434Crossref PubMed Scopus (379) Google Scholar, Yvon and Wadsworth, 2000Yvon A.M. Wadsworth P. Region-specific microtubule transport in motile cells.J. Cell Biol. 2000; 151: 1003-1012Crossref PubMed Scopus (37) Google Scholar). More recently, dual-wavelength fluorescent speckle microscopy has enabled visualization of MT coupling to retrograde actin flow in lamellae and also to anterograde actin movement within the cell body (Gupton et al., 2002Gupton S.L. Salmon W.C. Waterman-Storer C.M. Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells.Curr. Biol. 2002; 12: 1891-1899Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Salmon et al., 2002Salmon W.C. Adams M.C. Waterman-Storer C.M. Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells.J. Cell Biol. 2002; 158: 31-37Crossref PubMed Scopus (167) Google Scholar). Microtubules have also been shown to grow along F-actin bundles (Kodama et al., 2003Kodama A. Karakesisoglou I. Wong E. Vaezi A. Fuchs E. ACF7: An essential integrator of microtubule dynamics.Cell. 2003; 115: 343-354Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). One possible role for such coordinated actin-MT dynamics might be in mediating specific spatiotemporal regulation of FA dynamics to polarize cellular movements. Previous studies have demonstrated that MTs can specifically target peripheral FAs and promote their turnover, perhaps through MT motor-mediated delivery of key disassembly factors (Kaverina et al., 1998Kaverina I. Rottner K. Small J.V. Targeting, capture, and stabilization of microtubules at early focal adhesions.J. Cell Biol. 1998; 142: 181-190Crossref PubMed Scopus (248) Google Scholar, Kaverina et al., 1999Kaverina I. Krylyshkina O. Small J.V. Microtubule targeting of substrate contacts promotes their relaxation and dissociation.J. Cell Biol. 1999; 146: 1033-1044Crossref PubMed Scopus (371) Google Scholar, Krylyshkina et al., 2002Krylyshkina O. Kaverina I. Kranewitter W. Steffen W. Alonso M.C. Cross R.A. Small J.V. Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1.J. Cell Biol. 2002; 156: 349-359Crossref PubMed Scopus (97) Google Scholar, Krylyshkina et al., 2003Krylyshkina O. Anderson K.I. Kaverina I. Upmann I. Manstein D.J. Small J.V. Toomre D.K. Nanometer targeting of microtubules to focal adhesions.J. Cell Biol. 2003; 161: 853-859Crossref PubMed Scopus (131) Google Scholar). Additional explorations reveal roles for FAK and dynamin in MT-induced FA turnover, suggestive of involvement of actin dynamics and endocytosis (Ezratty et al., 2005Ezratty E.J. Partridge M.A. Gundersen G.G. Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase.Nat. Cell Biol. 2005; 7: 581-590Crossref PubMed Scopus (445) Google Scholar). The molecular nature that underlies the potentially crucial coordination of MT-actin cytoskeletons at sites of focal adhesions in mammalian cells remains elusive, but accumulating evidence suggests that MT plus ends (+tips) and +tip-tracking proteins may mediate this cytoskeletal crosstalk (Akhmanova and Steinmetz, 2008Akhmanova A. Steinmetz M.O. Tracking the ends: A dynamic protein network controls the fate of microtubule tips.Nat. Rev. Mol. Cell Biol. 2008; 9: 309-322Crossref PubMed Scopus (716) Google Scholar, Carvalho et al., 2003Carvalho P. Tirnauer J.S. Pellman D. Surfing on microtubule ends.Trends Cell Biol. 2003; 13: 229-237Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, Wu et al., 2006Wu X. Xiang X. Hammer 3rd, J.A. Motor proteins at the microtubule plus-end.Trends Cell Biol. 2006; 16: 135-143Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Although budding yeast lacks the major components of FAs, several tantalizing parallels exist between directed cell movement in mammalian cells and that which pulls and reorients the mitotic spindle along F-actin cables to its specific anchoring site within the yeast bud. In yeast, this intricate process requires an F-actin motor (Myo2) that binds to the adaptor protein Kar9, which in turn links indirectly to spindle MTs through the yeast homolog (Bim1) of the +tip protein EB1 (Kusch et al., 2003Kusch J. Liakopoulos D. Barral Y. Spindle asymmetry: A compass for the cell.Trends Cell Biol. 2003; 13: 562-569Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Yarm et al., 2001Yarm F. Sagot I. Pellman D. The social life of actin and microtubules: Interaction versus cooperation.Curr. Opin. Microbiol. 2001; 4: 696-702Crossref PubMed Scopus (33) Google Scholar). Unique to multicellular organisms, spectraplakins are broadly expressed and are unusual in their ability to bind directly to and crosslink MT and F-actin networks (Jefferson et al., 2004Jefferson J.J. Leung C.L. Liem R.K. Plakins: Goliaths that link cell junctions and the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2004; 5: 542-553Crossref PubMed Scopus (132) Google Scholar, Leung et al., 1999Leung C.L. Sun D. Zheng M. Knowles D.R. Liem R.K. Microtubule actin cross-linking factor (MACF): A hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.J. Cell Biol. 1999; 147: 1275-1286Crossref PubMed Scopus (171) Google Scholar, Sun et al., 2001Sun D. Leung C.L. Liem R.K. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): Identification of a novel group of microtubule associated proteins.J. Cell Sci. 2001; 114: 161-172Crossref PubMed Google Scholar, Yang et al., 1999Yang Y. Bauer C. Strasser G. Wollman R. Julien J.P. Fuchs E. Integrators of the cytoskeleton that stabilize microtubules.Cell. 1999; 98: 229-238Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Genetic studies in lower eukaryotes suggest that their functions might be especially prominent in the epidermis and nervous system (Bosher et al., 2003Bosher J.M. Hahn B.S. Legouis R. Sookhareea S. Weimer R.M. Gansmuller A. Chisholm A.D. Rose A.M. Bessereau J.L. Labouesse M. The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces.J. Cell Biol. 2003; 161: 757-768Crossref PubMed Scopus (108) Google Scholar, Jefferson et al., 2004Jefferson J.J. Leung C.L. Liem R.K. Plakins: Goliaths that link cell junctions and the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2004; 5: 542-553Crossref PubMed Scopus (132) Google Scholar, Roper et al., 2002Roper K. Gregory S.L. Brown N.H. The ‘spectraplakins’: Cytoskeletal giants with characteristics of both spectrin and plakin families.J. Cell Sci. 2002; 115: 4215-4225Crossref PubMed Scopus (135) Google Scholar). Of the mammalian spectraplakins, ACF7/MACF1 (actin crosslinking family 7/microtubule and actin crosslinking factor 1) is expressed and localized in patterns that most closely resemble Drosophila spectraplakin. Although preimplantation lethality precludes in vivo analysis, visceral endoderm cells derived from cultured ACF7 null blastocysts reveal defects in the coordination of MT and actin cytoskeletons, accompanied by skewed cytoplasmic trajectories and altered dynamic instability of MTs. ACF7-deficient cells also fail to maintain cellular polarity in scratch-wounded cultures (Kodama et al., 2003Kodama A. Karakesisoglou I. Wong E. Vaezi A. Fuchs E. ACF7: An essential integrator of microtubule dynamics.Cell. 2003; 115: 343-354Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In the present report, we employ conditional gene targeting to ablate ACF7 expression in skin of mice. Our findings have uncovered key roles for ACF7 in wound healing and epidermal migration, which we trace to an ability of ACF7 to target MTs that preferentially track along F-actin to FAs. By uncoupling this connection, we show that it is essential for controlling FA assembly and dynamics. Surprisingly, however, we find that the MT, F-actin, and +tip protein binding capabilities of ACF7 are not sufficient to rescue all of the functions of wild-type (WT) ACF7. Subsequently, we've identified an ∼3000 residue central domain that bears sequence similarity to the Smc family of ATPases. We show that ACF7 can function as an ATPase, and, importantly, it behaves as an actin-regulated rather than MT- or calcium-regulated ATPase. Hitherto unrecognized in the spectraplakin family, this activity unveils important mechanistic insights into how spectraplakins evolved to function as unique macromolecular integrators that regulate dynamic actin-MT-mediated processes and how ACF7 in particular functions in establishing and maintaining proper cytoskeletal coordination during cell movement. We conditionally targeted ACF7 by inserting two loxP sites flanking exon 6 and exon 7 (Figure S1 available online), which reside within the conserved plakin domain common to all known isoforms of ACF7 and other spectraplakins (Bernier et al., 2000Bernier G. Pool M. Kilcup M. Alfoldi J. De Repentigny Y. Kothary R. Acf7 (MACF) is an actin and microtubule linker protein whose expression predominates in neural, muscle, and lung development.Dev. Dyn. 2000; 219: 216-225Crossref PubMed Scopus (50) Google Scholar, Jefferson et al., 2004Jefferson J.J. Leung C.L. Liem R.K. Plakins: Goliaths that link cell junctions and the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2004; 5: 542-553Crossref PubMed Scopus (132) Google Scholar, Karakesisoglou et al., 2000Karakesisoglou I. Yang Y. Fuchs E. An epidermal plakin that integrates actin and microtubule networks at cellular junctions.J. Cell Biol. 2000; 149: 195-208Crossref PubMed Scopus (135) Google Scholar, Leung et al., 1999Leung C.L. Sun D. Zheng M. Knowles D.R. Liem R.K. Microtubule actin cross-linking factor (MACF): A hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons.J. Cell Biol. 1999; 147: 1275-1286Crossref PubMed Scopus (171) Google Scholar, Sun et al., 2001Sun D. Leung C.L. Liem R.K. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): Identification of a novel group of microtubule associated proteins.J. Cell Sci. 2001; 114: 161-172Crossref PubMed Google Scholar). Their deletion is predicted to shift the coding sequence of downstream exons. Mice homozygous for the germline floxed (flanking loxP) insertions were bred to K14-Cre recombinase transgenic mice, which efficiently excised floxed exons by embryonic day E15.5 (Vasioukhin et al., 1999Vasioukhin V. Degenstein L. Wise B. Fuchs E. The magical touch: Genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin.Proc. Natl. Acad. Sci. USA. 1999; 96: 8551-8556Crossref PubMed Scopus (442) Google Scholar) (Figure 1A). Neonatal mice genotypic for K14-Cre and ACF7fl/fl alleles (conditional knockout, cKO) were born in the expected Mendelian numbers and grew to normal size as adults. Immunoblot analyses with an antibody specific for all known ACF7 isoforms confirmed the reduction of protein in heterozygous animal skin epidermis (K14-Cre: ACF7fl/+) and absence of ACF7 in cKO skin epidermis (Figure 1B). This was further verified by immunofluorescence, which documented the absence of ACF7 in epidermis and hair follicles (Figure 1C). Although the targeting could theoretically permit expression of a truncated N-terminal ACF7, immunoblot with an antibody recognizing the N-terminal sequence of ACF7 did not detect any low-molecular-weight protein product in KO keratinocyte lysate (Figure S2A), suggesting that the truncated protein was not expressed or unstable. Surprisingly, ACF7 cKO animals showed no gross morphological changes in skin or hair coat. Histologically, epidermal homeostasis appeared normal, and immunofluorescence with antibodies against β4 integrin (basal surface of the epidermis), keratin 5 (basal layer), keratin 10 (spinous layer), and loricrin (granular layer) all displayed localization patterns analogous to WT skin (Figure S2B). However, when challenged to respond to injury, ACF7 cKO skin exhibited a significant delay in repairing full-thickness wounds. Histological analysis and quantification revealed that the area of hyperproliferative epithelium (HE) that typically proliferates and migrates into the wound site was diminished by more than 30% over 2–4 days after injury. (Figures S2C and S2D, Figure 1D). Interestingly, despite the delay in wound closure, no significant decrease was found in proliferation or apoptosis, as judged by labeling for phospho-histone H3, Ki67, or activated caspase 3, respectively (Figures S2D and S2E and data not shown). Rather, the delayed wound response in ACF7-deficient skin seemed more likely to be rooted in alterations in cell migration, which we documented by culturing primary keratinocytes (1°MK) from cKO and littermate control skins and monitoring the polarized rate of cell migration during recovery of ∼500 μm scratches introduced into keratinocyte monolayers. Whereas WT 1°MK closed the gap within 36 hr, KO keratinocytes moved only ∼20% of this distance into the gap (Figures 1E and 1F). As expected from our in vivo data, growth curve experiments showed no significant difference between KO and WT cells (Figure 1F). Videomicroscopy permitted imaging and monitoring of the velocities and directed migration of individual keratinocytes. Assays of representative 1°MK highlighted the significantly slower movements of KO cells compared to their WT counterparts (Movie S1). Quantification of these movements revealed an ∼60% decrease in average speed on fibronectin (FN) (Figures 1G and 1H). In contrast, when kymography was used to record membrane dynamics (Lauffenburger and Horwitz, 1996Lauffenburger D.A. Horwitz A.F. Cell migration: A physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3142) Google Scholar), no differences were detected in either duration of protrusion or protrusion rate (Figures S3A and S3B). To explore other possible mechanisms that might underlie ACF7 regulation of cell migration, we examined the contribution of cell/ECM adhesion to cell migration after ACF7 loss. To manipulate cell adhesive strength, we plated 1°MK onto dishes coated with FN at different concentrations (Gupton and Waterman-Storer, 2006Gupton S.L. Waterman-Storer C.M. Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.Cell. 2006; 125: 1361-1374Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, Palecek et al., 1997Palecek S.P. Loftus J.C. Ginsberg M.H. Lauffenburger D.A. Horwitz A.F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.Nature. 1997; 385: 537-540Crossref PubMed Scopus (1137) Google Scholar) and then recorded cell migration by videomicroscopy. Interestingly, by simply reducing the amount of underlying matrix protein, we were able to largely equalize the marked differences in velocities displayed by KO versus WT cells (Figure 2A). These data suggested that ACF7 null 1°MK migrated aberrantly because they were defective in how they adhered to their underlying substratum. Immunofluorescence microscopy revealed enhanced labeling of FAs in ACF7 null 1°MK relative to WT controls (Figure 2B). Notably, the robust FAs in cells lacking ACF7 were also closely associated with cables of F-actin (stress fibers). Quantification documented a substantial increase in both size and total fluorescence intensity of FAs in KO 1°MK. Figure 2C shows the quantifications for vinculin, a representative structural component of FAs. FA enlargement at the cell periphery is a sign of defects in FA dynamics, which are essential for cells to move (Berrier and Yamada, 2007Berrier A.L. Yamada K.M. Cell-matrix adhesion.J. Cell. Physiol. 2007; 213: 565-573Crossref PubMed Scopus (655) Google Scholar, Burridge and Chrzanowska-Wodnicka, 1996Burridge K. Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signaling.Annu. Rev. Cell Dev. Biol. 1996; 12: 463-518Crossref PubMed Scopus (1612) Google Scholar, Delon and Brown, 2007Delon I. Brown N.H. Integrins and the actin cytoskeleton.Curr. Opin. Cell Biol. 2007; 19: 43-50Crossref PubMed Scopus (164) Google Scholar, Lauffenburger and Horwitz, 1996Lauffenburger D.A. Horwitz A.F. Cell migration: A physically integrated molecular process.Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3142) Google Scholar, Ridley et al., 2003Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Cell migration: Integrating signals from front to back.Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3584) Google Scholar). To explore the nature of this defect in ACF7 null 1°MK, we began by employing confocal videomicroscopy to trace the behavior of individual FAs (Webb et al., 2004Webb D.J. Donais K. Whitmore L.A. Thomas S.M. Turner C.E. Parsons J.T. Horwitz A.F. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.Nat. Cell Biol. 2004; 6: 154-161Crossref PubMed Scopus (1010) Google Scholar). To monitor the process, we transfected cells with plasmids encoding either DsRed-Zyxin or GFP-Paxillin (Kodama et al., 2003Kodama A. Karakesisoglou I. Wong E. Vaezi A. Fuchs E. ACF7: An essential integrator of microtubule dynamics.Cell. 2003; 115: 343-354Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, Schober et al., 2007Schober M. Raghavan S. Nikolova M. Polak L. Pasolli H.A. Beggs H.E. Reichardt L.F. Fuchs E. Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics.J. Cell Biol. 2007; 176: 667-680Crossref PubMed Scopus (175) Google Scholar). Examples of the perturbations in FA dynamics arising from ACF7 deficiency are shown in complete form in Movie S2, and in montages are shown in Figure 3A. Most strikingly, in a 120 min interval of observation, FAs in KO cells were often static, whereas the majority of FAs in WT keratinocytes underwent continual bouts of formation, maturation, and disassembly. Quantifications of the kinetics of hundreds of individual FAs revealed a significant and marked decrease in both the assembly and disassembly rates of FAs in KO cells (Figure 3B). This was interesting, as FAK KO keratinocytes display defects in disassembly but not assembly rates (Schober et al., 2007Schober M. Raghavan S. Nikolova M. Polak L. Pasolli H.A. Beggs H.E. Reichardt L.F. Fuchs E. Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics.J. Cell Biol. 2007; 176: 667-680Crossref PubMed Scopus (175) Google Scholar). The defects in FA dynamics were further substantiated by fluorescence recovery after photobleaching (FRAP) experiments (Figure 3C). By comparing GFP-Paxillin (Pxn)-transfected 1°MK, we found that ACF7 deficiency resulted in a strong decrease in the fraction of mobile protein and a significant increase in half-times of fluorescence recovery after bleaching (Figure 3D). Together, these data provide compelling evidence that FAs are more stable in the absence of ACF7, suggesting that ACF7 regulates cell migration at least in part by promoting FA dynamics. Rho GTPases and their control of actomyosin activity are known to play a pivotal role in regulating FA dynamics and cell migration (Jaffe and Hall, 2005Jaffe A.B. Hall A. Rho GTPases: Biochemistry and biology.Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2199) Google Scholar). However, biochemical pull-down assays revealed comparable overall levels of the GTP-bound state of RhoA, Rac1, and Cdc42 in WT and ACF7 KO cells (Figures 3E and 3F). Similarly, no significant differences were detected in myosin light chain Ser19 phosphorylation, which correlates with myosin contraction activity (Figures S4A and S4B). FA turnover typically involves a signaling pathway centering on FAK and Src tyrosine kinases, which are activated upon integrin-ECM engagement (Guan, 1997Guan J.L. Focal adhesion kinase in integrin signaling.Matrix Biol. 1997; 16: 195-200Crossref PubMed Scopus (126) Google Scholar, Mitra et al., 2005Mitra S.K. Hanson D.A. Schlaepfer D.D. Focal adhesion kinase: In command and control of cell motility.Nat. Rev. Mol. Cell Biol. 2005; 6: 56-68Crossref PubMed Scopus (1796) Google Scholar). However, ACF7 null 1°MK exhibited no obvious change in either total FAK or Y397 phosphorylated (active) FAK (Guan, 1997Guan J.L. Focal adhesion kinase in integrin signaling.Matrix Biol. 1997; 16: 195-200Crossref PubMed Scopus (126) Google Scholar, Parsons, 2003Parsons J.T. Focal adhesion kinase: The first ten years.J. Cell Sci. 2003; 116: 1409-1416Crossref PubMed Scopus (1089) Google Scholar) (Figure 3G). Moreover, irrespective of ACF7 status, Y397 phosphorylation was abolished when cells were placed in suspension, suggesting that the dependency of activated integrins for FAK activity was not perturbed by loss of ACF7 function. Consistent with this notion, Src activity was unchanged as determined by phosphorylation levels of Y418 Src (Figure 3G). In suspension, WT and KO cells displayed similar decreases in Src activity. These findings were in good agreement with our kinetic studies and provided additional evidence that the impaired FA dynamics caused by ACF7 deficiency cannot be explained by alterations in the overall activity of Rho GTPases or FAK/Src signaling. Given the absence of changes in Rho/FAK/Src activities in ACF7-null 1°MK and increasing evidence that MT targeting is involved in FA turnover (Kaverina et al., 1999Kaverina I. Krylyshkina O. Small J.V. Microtubule targeting of substrate contacts promotes their relaxation and dissociation.J. Cell Biol. 1999; 146: 1033-1044Crossref PubMed Scopus (371) Google Scholar, Rodriguez et al., 2003Rodriguez O.C. Schaefer A.W. Mandato C.A. Forscher P. Bement W.M. Waterman-Storer C.M. Conserved microtubule-actin interactions in cell movement and morphogenesis.Nat. Cell Biol. 2003; 5: 599-609Crossref PubMed Scopus (681) Google Scholar), we turned to the possibility that coordinated cytoskeletal dynamics and/or targeting of MT to FAs might be responsible for the defects in FA dynamics caused by loss-of-function mutations in ACF7. Interestingly, treatment of nocodazole, a MT-depolymerizing drug, dramatically reduced the rate of FA turnover in WT 1°MK to a similar level as in untreated KO cells. On the contrary, the same treatment did not further inhibit FA turnover in KO keratinocytes, despite their comparable sensitivity to the drug and comparable overall levels of polymerized tubulin (Figure 3H, Figure S5). These data suggested that ACF7's effect on FA disassembly in epidermal cells is mediated by polymerized MT networks. Immunofluorescence microscopy unveiled additional defects in KO MT networks. In contrast to WT keratinocytes, where MTs bundles projected radially toward the cell periphery and paralleled the organization of actin stress fibers, MTs in ACF7 null keratinocytes were often curled and bent and no longer coaligned with actin cables (Figure 4A). Although the curly, bent behavior of MTs was remarkably similar to that observed in ACF7 null endodermal cells (Kodama et al., 2003Kodama A. Karakesisoglou I. Wong E. Vaezi A. Fuchs E. ACF7: An essential integrator of microtubule dynamics.Cell. 2003; 115: 343-354Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), the robust network of stress fibers and FAs in keratinocytes accentuated the extent to which coordination between actin and MT networks was disrupted in the absence of ACF7. The prominent actin-FA network in keratinocytes led us to discover a striking and important perturbation in MT dynamics that had not been noted previously, namely that the MT ends that normally converge at peripheral FAs were missing in ACF7 null cells (Figure 4B). Since ACF7 localized to these sites in WT keratinocytes (Figure 4B), we tested whether ACF7 might physically interact with +tip proteins, as has been found for two other spectraplakins (Kakapo and BPAG1) (Slep et al., 2005Slep K.C. Rogers S.L. Elliott S.L. Ohkura H. Kolodziej P.A. Vale R.D. Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end.J. Cell Biol. 2005; 168: 587-598Crossref PubMed Scopus (143) Google Scholar, Subramanian et al., 2003Subramanian A. Prokop A. Yamamoto M. Sugimura K. Uemura T. Betschinger J. Knoblich J.A. Volk T. Shortstop recruits EB1/APC1 and promotes microtubule assembly at the muscle-tendon junction.Curr. Biol. 2003; 13: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). As shown in Figure 4C, keratinocyte ACF7 specifically coimmunoprecip" @default.
• W2086681436 created "2016-06-24" @default.
• W2086681436 creator A5014504040 @default.
• W2086681436 creator A5044733394 @default.
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• W2086681436 date "2008-10-01" @default.
• W2086681436 modified "2023-10-03" @default.
• W2086681436 title "ACF7 Regulates Cytoskeletal-Focal Adhesion Dynamics and Migration and Has ATPase Activity" @default.
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