Local Myo9b RhoGAP activity regulates cell motility

To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension, whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase-activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60–derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration. To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension, whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase-activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60–derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration. Cell function is tightly coupled with cell morphology, and coordinated alterations in cell morphology are a prerequisite for cell translocation. Cell migration depends on a polarized morphology with a protruding front and a retracting back (1Tschumperlin D.J. Fibroblasts and the ground they walk on.Physiology (Bethesda). 2013; 28: 380-390Crossref PubMed Scopus (62) Google Scholar, 2Trepat X. Chen Z. Jacobson K. Cell migration.Compr. Physiol. 2012; 2: 2369-2392Crossref PubMed Scopus (143) Google Scholar). Key regulators of these two opposing activities are small monomeric GTPases of the Rho subfamily (3Jaffe A.B. Hall A. Rho GTPases: biochemistry and biology.Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2200) Google Scholar). They regulate the dynamics and organization of the actin cytoskeleton. Rho proteins are activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP and inactivated by GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis, switching the GTPase back to the inactive GDP-bound state. A third class of proteins, named GDP dissociation inhibitors, is sequestering the GTPases in the cytosol. Activation of RhoA increases filamentous actin concentration and myosin II contractility (3Jaffe A.B. Hall A. Rho GTPases: biochemistry and biology.Annu. Rev. Cell Dev. Biol. 2005; 21: 247-269Crossref PubMed Scopus (2200) Google Scholar). Acto-myosin II contractility at the sides and back of migrating cells pushes the nucleus forward and retracts the rear (4Symons M. Segall J.E. Rac and Rho driving tumor invasion: who's at the wheel?.Genome Biol. 2009; 10: 213Crossref PubMed Scopus (43) Google Scholar, 5Petrie R.J. Yamada K.M. At the leading edge of three-dimensional cell migration.J. Cell Sci. 2012; 125: 5917-5926Crossref PubMed Scopus (198) Google Scholar, 6Paul C.D. Hung W.C. Wirtz D. Konstantopoulos K. Engineered models of confined cell migration.Annu. Rev. Biomed. Eng. 2016; 18: 159-180Crossref PubMed Scopus (50) Google Scholar). Cell protrusion is mainly driven by Rac- and Cdc42-induced actin polymerization (7Krause M. Gautreau A. Steering cell migration: lamellipodium dynamics and the regulation of directional persistence.Nat. Rev. Mol. Cell Biol. 2014; 15: 577-590Crossref PubMed Scopus (291) Google Scholar). However, FRET biosensors monitoring RhoA activity indicated an enhanced RhoA activity at the protruding membrane, but the functional significance of this finding is not understood (8Kurokawa K. Nakamura T. Aoki K. Matsuda M. Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells.Biochem. Soc. Trans. 2005; 33: 631-634Crossref PubMed Scopus (59) Google Scholar, 9Pertz O. Hodgson L. Klemke R.L. Hahn K.M. Spatiotemporal dynamics of RhoA activity in migrating cells.Nature. 2006; 440: 1069-1072Crossref PubMed Scopus (583) Google Scholar, 10Machacek M. Hodgson L. Welch C. Elliott H. Pertz O. Nalbant P. Abell A. Johnson G.L. Hahn K.M. Danuser G. Coordination of Rho GTPase activities during cell protrusion.Nature. 2009; 461: 99-103Crossref PubMed Scopus (647) Google Scholar, 11Hinde E. Digman M.A. Hahn K.M. Gratton E. Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 135-140Crossref PubMed Scopus (50) Google Scholar). Regulatory networks capable of self-organizing cell polarization have been identified, including positive feedback mechanisms, mutual inhibition, and inhibition with positive feedback (12Chau A.H. Walter J.M. Gerardin J. Tang C. Lim W.A. Designing synthetic regulatory networks capable of self-organizing cell polarization.Cell. 2012; 151: 320-332Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The signaling networks regulating the front and back have also been shown to reinforce each other (13Van Keymeulen A. Wong K. Knight Z.A. Govaerts C. Hahn K.M. Shokat K.M. Bourne H.R. To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front.J. Cell Biol. 2006; 174: 437-445Crossref PubMed Scopus (125) Google Scholar, 14Ku C.-J. Wang Y. Weiner O.D. Altschuler S.J. Wu L.F. Network crosstalk dynamically changes during neutrophil polarization.Cell. 2012; 149: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To understand cell migration on a molecular level, the molecules controlling subcellular RhoA signaling circuits and their linkage with Rac1 signaling need to be determined. Many regulators of Rho and Rac signaling, as well as effector molecules, have been identified. The task is now to assign cellular functions to all of them and to integrate them into physiological circuits. The RhoGAP myosin IXb (Myo9b) accumulates in protrusive cellular structures containing dynamic actin filaments owing to its actin-based motor activity (15van den Boom F. Düssmann H. Uhlenbrock K. Abouhamed M. Bähler M. The myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.Mol. Biol. Cell. 2007; 18: 1507-1518Crossref PubMed Scopus (0) Google Scholar). Deletion of Myo9b causes impaired cell migration both in vitro and in vivo (16Hanley P.J. Xu Y. Kronlage M. Grobe K. Schön P. Song J. Sorokin L. Schwab A. Bähler M. Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 12145-12150Crossref PubMed Scopus (74) Google Scholar, 17Chandhoke S.K. Mooseker M.S. A role for myosin IXb, a motor-RhoGAP chimera, in epithelial wound healing and tight junction regulation.Mol. Biol. Cell. 2012; 23: 2468-2480Crossref PubMed Scopus (31) Google Scholar, 18Xu Y. Pektor S. Balkow S. Hemkemeyer S.A. Liu Z. Grobe K. Hanley P.J. Shen L. Bros M. Schmidt T. Bähler M. Grabbe S. Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.J. Immunol. 2014; 192: 3559-3568Crossref PubMed Scopus (32) Google Scholar, 19Yi F. Kong R. Ren J. Zhu L. Lou J. Wu J.Y. Feng W. Noncanonical Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a dual-arginine-finger mechanism.J. Mol. Biol. 2016; 428: 3043-3057Crossref PubMed Scopus (10) Google Scholar, 20Moalli F. Ficht X. Germann P. Vladymyrov M. Stolp B. de Vries I. Lyck R. Balmer J. Fiocchi A. Kreutzfeldt M. Merkler D. Iannacone M. Ariga A. Stoffel M.H. Sharpe J. et al.The rho regulator myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells.J. Exp. Med. 2018; 215: 1869-1890Crossref PubMed Scopus (12) Google Scholar). We hypothesized that Myo9b is recruited to extending lamellipodia through Rac-induced actin polymerization to locally inhibit RhoA activity at the leading edge. Local inhibition of RhoA activity by Rac activity could prevent contractility and stabilize a positive feedback loop supporting protrusion. To address this hypothesis that Myo9b acts locally, we firstly tested whether Rac activation is sufficient for the recruitment of Myo9b to protruding lamellipodia. Secondly, in HL-60 macrophages, we replaced endogenous Myo9b with Myo9b mutants lacking either GAP or motor activity and subsequently characterized the motility of these genetically modified cells. We show here that Rac activity is sufficient for Myo9b recruitment to lamellipodial protrusions and that local recruitment of Myo9b RhoGAP activity is important for directional cell migration. Myo9b motor activity directs Myo9b to dynamic actin filament networks that drive lamellipodial protrusion (15van den Boom F. Düssmann H. Uhlenbrock K. Abouhamed M. Bähler M. The myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.Mol. Biol. Cell. 2007; 18: 1507-1518Crossref PubMed Scopus (0) Google Scholar). How Myo9b motor activity and subsequent accumulation in protruding lamellipodia is regulated is not known. To test if Rac-induced signaling and ensuing actin polymerization would be sufficient for the recruitment of Myo9b, we transfected NIH3T3 cells with photoactivatable Rac1 (PA-Rac1). Local photoactivation of PA-Rac1 induced protrusive lamellipodia (Fig. 1, A–C). Cells cotransfected with either mCherry-Myo9b-WT or mCherry-Myo9b-R1695M, a mutant lacking RhoGAP activity, showed that both constructs accumulate at the front of those protruding lamellipodia (Fig. 1, A–C). These Myo9b constructs localized similarly to the dynamic actin filaments that drive lamellipodia protrusion as monitored by Lifeact-mRFPruby. The recruitment of Myo9b to the leading edge required its motor activity. Two motor mutants that are defective in either nucleotide binding (21Bejsovec A. Anderson P. Functions of the myosin ATP and actin binding sites are required for C. elegans thick filament assembly.Cell. 1990; 60: 133-140Abstract Full Text PDF PubMed Scopus (58) Google Scholar) or hydrolysis (22Shimada T. Sasaki N. Ohkura R. Sutoh K. Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II.Biochemistry. 1997; 36: 14037-14043Crossref PubMed Scopus (75) Google Scholar, 23Furch M. Fujita-Becker S. Geeves M.A. Holmes K.C. Manstein D.J. Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin.J. Mol. Biol. 1999; 290: 797-809Crossref PubMed Scopus (68) Google Scholar) were not recruited to the leading edge of protruding lamellipodia and localized comparable to the cytosolic protein mCherry (Fig. 1, A–C). These results show that Rac-induced lamellipodia formation leads to the recruitment of Myo9b and that its motor activity is essential for this purpose. Myeloid HL-60 cells express Myo9b and can be differentiated into macrophages (Fig. 2C and Fig. 3). In contrast to primary macrophages, they can be genetically modified and subsequently propagated. To study the function of Myo9b in HL-60–derived macrophages, we disrupted the Myo9b alleles using CRISPR/Cas9. As schematically shown in Figure 2 A, two double-strand breaks were induced by four specific gRNA sequences and a Cas9 nickase. Selected cell clones were screened by PCR (Fig. 2B). The individual Myo9b alleles of potential Myo9b knockout cell clones were further analyzed by sequencing, which confirmed the insertion of nonsense mutations or deletion of nucleotides (Fig. S1). In accordance with the sequencing results, the expression of the Myo9b protein was abrogated in the corresponding cell clones as shown in Figure 2C. Cell clones that were not modified by CRISPR/Cas9 served as WT-like controls. WT and Myo9b-deficient HL-60 cells were differentiated into macrophages. In WT HL-60 cells Myo9b expression was not obviously altered upon cell differentiation (Fig. 3A). Differentiation was monitored by the expression of the marker protein CD11b. Irrespective of Myo9b expression, the differentiation marker CD11b was induced to the same extent upon HL-60 macrophage differentiation (Fig. 3, B–D). Having established that macrophage differentiation is not affected by Myo9b expression, we explored whether Myo9b regulates the morphology of adherent HL-60 macrophages. Therefore, we determined the following three parameters indicative of cell morphology: the area, the circularity index, and the aspect ratio of a fitted ellipse (Fig. 4). Cells from three different Myo9b-deficient clones covered a significantly smaller surface area than cells from WT clones, suggestive of a more contracted state (Fig. 4, A–B). This was further reflected in a higher circularity index for the Myo9b-deficient cells than in cells from WT clones (Fig. 4C) and a lower aspect ratio of a fitted ellipse (Fig. 4D).Figure 3Myo9b does not influence HL-60 cell differentiation to macrophages. A, Myo9b protein expression increases slightly during PMA-induced HL-60 macrophage differentiation (w/o: undifferentiated). B, flow cytometry analysis of the cell surface expression of differentiation marker CD11b in undifferentiated and differentiated HL-60 cells. No differences in the upregulation of expression were detected between wild-type and Myo9b-deficient cells. Histograms of a representative experiment are shown for a wild-type and a Myo9b-deficient cell line. C–D, quantification of CD11b-positive cells of indicated cell lines that were either undifferentiated (C) or differentiated with PMA for 1 day (D). Data are from four independent experiments, n.s., not significant by the Kruskal–Wallis test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Loss of Myo9b alters HL-60 macrophage cell morphology. A, HL-60 cells differentiated to macrophages in fibronectin-coated plates display a different cell morphology subject to whether they express Myo9b (48 (WT)) or not (57 (KO)). Scale bar, 20 μm. Detailed analysis of cells revealed that the Myo9b-deficient cells not only cover a smaller surface area (B) but also are less well polarized as indicated by an increased circularity index (C) and a lower aspect ratio of a filled ellipse (D). n = 17 to 20 cells per cell clone. A p-value of p ≥ 0.05 was regarded as not significant (n.s.), p ≤ 0.05 as a trend (∗), p ≤ 0.01 as significant (∗∗), and p ≤ 0.001 as highly significant (∗∗∗).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we tracked the two-dimensional migration of HL-60 macrophages plated on fibronectin (Fig. 5A). WT-like macrophages from clones 48 and 54 migrated with a faster average velocity than macrophages from the three different Myo9b-deficient clones 4, 57, and 72 (Fig. 5B). Analysis of the directionality of migration revealed some variability between Myo9b-deficient clones. Nevertheless, macrophages from the two WT-like clones migrated significantly more directed than macrophages from the Myo9b-deficient clones 4 and 72 (Fig. 5C). Cells from the Myo9b-deficient clone 57 showed somewhat variable directionality, but on average, cells were still less directed than cells of the two WT-like clones (Fig. 5C). To test whether the altered morphology, decreased migration velocity, and directionality of Myo9b-deficient HL-60 macrophages can be rescued by the expression of Myo9b-EGFP constructs, we created cell lines of the Myo9b-deficient cell clones 57 and 72 that stably express either Myo9bWT-EGFP or just EGFP alone as a control. Furthermore, to analyze whether the phenotypes observed in Myo9b-deficient HL-60 cells are due to global or local regulation of Rho activity by Myo9b-RhoGAP, we expressed Myo9b constructs exhibiting point mutations that abrogate either its RhoGAP activity (Myo9bRM GAP−-EGFP) or its motor activity (Myo9bGR nucleotide−-EGFP and Myo9bRC hydrolysis−-EGFP (Fig. 6). The expression levels of the different Myo9b-mutant constructs varied somewhat (Fig. 6, B–E). Cells derived from the Myo9b-deficient clone 57 expressed Myo9bWT-EGFP and Myo9bRM GAP−-EGFP to comparable levels of endogenous Myo9b in WT cells. The two different Myo9b constructs predicted to lack motor activity were expressed at roughly half the amount of the other two aforementioned constructs (Fig. 6, B, D and E). In contrast, cell clones derived from the Myo9b-deficient clone 72 expressed Myo9bWT-EGFP to one-quarter of the endogenous Myo9b level, whereas Myo9bRM GAP−-EGFP was expressed at somewhat higher amounts of endogenous Myo9b levels (Fig. 6, C–E). Further analysis of the cell clones that express Myo9b-EGFP constructs by fluorescence microscopy revealed that individual cells of a given clone expressed their respective constructs at comparable levels (Fig. S2). Furthermore, the Myo9b WT and GAP−-EGFP constructs were enriched at the leading edge of protruding lamellipodia, whereas the two Myo9b motor mutant constructs nucleotide−- and hydrolysis−-EGFP were not and showed the same localization as EGFP alone (Fig. S2). First, we analyzed the ability of different Myo9b constructs to rescue the cell morphology of Myo9b-deficient cells adherent to a 2D surface. The expression of Myo9b-EGFP in Myo9b-deficient cells of clones 57 and 72 rescued cell spreading as shown by an increase in surface area covered by the cells, whereas cells that solely expressed EGFP still covered a smaller area (Fig. 7, A–B). It is noteworthy that expression of one-quarter amount of the endogenous Myo9b in the cells derived from clone 72 was sufficient to rescue the cell spreading phenotype. In agreement with the assumption that the smaller surface area covered by Myo9b-deficient cells is a result of higher RhoA activity, the expression of RhoGAP-inactive Myo9bRM-EGFP did not increase cell area even when expressed at twice the endogenous Myo9b level (Fig. 7, A–B). The nucleotide-binding motor mutant Myo9bGR-EGFP that does not accumulate at the leading edge also did not increase cell area. Of note, expression of the predicted ATP hydrolysis motor mutant Myo9bRC-EGFP increased cell area similar to WT Myo9b-EGFP (Fig. 7, A–B), indicating that cell area might be affected by evenly distributed diffusible RhoGAP activity. Analysis of the circularity index revealed that expression of WT Myo9b in the Myo9b null background, but not EGFP, lowered the value substantially to a level comparable to that observed with WT cells. Myo9b RhoGAP mutant as well as both motor mutants lowered the circularity index only modestly, if at all (Fig. 7C). Comparable results were obtained for the aspect ratio of a fitted ellipse. It increased to levels of WT cells when WT Myo9b was expressed, but only slightly increased upon expression of Myo9b RhoGAP or motor mutants (Fig. 7D). Next, we analyzed whether the expression of different Myo9b constructs in Myo9b-deficient HL-60 macrophages affects not only their morphology but also their migration. The expression of EGFP-labeled rat Myo9b in Myo9b-deficient cells derived from either clone 57 or clone 72 increased migration velocity and directionality to WT cell levels. However, expression of EGFP did not rescue the reduced velocity and directionality (Fig. 8). Rescue of the migration phenotype depended on the RhoGAP activity of Myo9b. Expression of a RhoGAP-deficient Myo9b-EGFP mutant barely affected velocity and directionality of migration (Fig. 8). In Myo9b-deficient cells from both clones 57 and 72, velocity and directionality of migration were not significantly altered upon expression of a RhoGAP-inactive Myo9b mutant (Fig. 8, A–D). To probe for potential involvement of the motor activity of Myo9b in regulating cell migration, we analyzed the migration of cells that express either of two motor mutant constructs that are predicted to block nucleotide binding (Myo9bG244R) and ATP hydrolysis (Myo9bR295C), respectively (Fig. 6, A–B). Expression of Myo9bGR nucleotide−-EGFP increased neither velocity nor directionality of the cells (Fig. 8, A and C), indicating that motor activity is important. Similarly, the expression of the second Myo9bRC hydrolysis−-EGFP motor mutant did not yield cells that migrated faster or more directional than the original Myo9b-deficient cells (Fig. 8, A and C). These results suggest that proper positioning of the functional RhoGAP domain by the motor activity regulates cell migration and directionality. Myo9b activates specifically the GTP hydrolysis of RhoA and thereby switches it to its inactive GDP-bound state (24Reinhard J. Scheel A.A. Diekmann D. Hall A. Ruppert C. Bähler M. A novel type of myosin implicated in signalling by rho family GTPases.EMBO J. 1995; 14: 697-704Crossref PubMed Scopus (147) Google Scholar, 25Müller R.T. Honnert U. Reinhard J. Bähler M. The rat myosin myr 5 is a GTPase-activating protein for rho in vivo: essential role of arginine 1695.Mol. Biol. Cell. 1997; 8: 2039-2053Crossref PubMed Scopus (74) Google Scholar). In leucocytes, the RhoGAP activity of Myo9b regulates cell morphology and migration (16Hanley P.J. Xu Y. Kronlage M. Grobe K. Schön P. Song J. Sorokin L. Schwab A. Bähler M. Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 12145-12150Crossref PubMed Scopus (74) Google Scholar, 18Xu Y. Pektor S. Balkow S. Hemkemeyer S.A. Liu Z. Grobe K. Hanley P.J. Shen L. Bros M. Schmidt T. Bähler M. Grabbe S. Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.J. Immunol. 2014; 192: 3559-3568Crossref PubMed Scopus (32) Google Scholar, 20Moalli F. Ficht X. Germann P. Vladymyrov M. Stolp B. de Vries I. Lyck R. Balmer J. Fiocchi A. Kreutzfeldt M. Merkler D. Iannacone M. Ariga A. Stoffel M.H. Sharpe J. et al.The rho regulator myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells.J. Exp. Med. 2018; 215: 1869-1890Crossref PubMed Scopus (12) Google Scholar). But so far, it was not known whether Myo9b regulates cell morphology and migration by accelerating Rho GTPase activity in cells globally or locally. Here we show that Myo9b controls cell morphology and migration by negatively regulating Rho activity locally at sites of actin polymerization such as at the leading edge of migrating cells. We have reported previously that Myo9b accumulates at the leading edge of protruding lamellipodia together with elongating actin filaments that drive protrusion. Prerequisite for this accumulation was a functional motor domain of Myo9b (15van den Boom F. Düssmann H. Uhlenbrock K. Abouhamed M. Bähler M. The myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.Mol. Biol. Cell. 2007; 18: 1507-1518Crossref PubMed Scopus (0) Google Scholar). Here we show that activation of Rac is sufficient to induce the relocalization of Myo9b to extending lamellipodia. Rac activation induces actin polymerization, and the motor domain of Myo9b may interact specifically with newly polymerizing actin filaments or networks. This possibility is supported by the observation that Myo9b also accumulates at filopodial tips (15van den Boom F. Düssmann H. Uhlenbrock K. Abouhamed M. Bähler M. The myosin IXb motor activity targets the myosin IXb RhoGAP domain as cargo to sites of actin polymerization.Mol. Biol. Cell. 2007; 18: 1507-1518Crossref PubMed Scopus (0) Google Scholar). Alternatively, the Myo9b motor could be switched from an inhibited to an active state by a Rac-dependent signaling pathway that acts in parallel to actin polymerization. For instance, multiple phosphorylation sites have been identified in Myo9b (26Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 27Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10762-10767Crossref PubMed Scopus (1171) Google Scholar, 28Huttlin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Beausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. A tissue-specific atlas of mouse protein phosphorylation and expression.Cell. 2010; 143: 1174-1189Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar, 29Gnad F. Gunawardena J. Mann M. PHOSIDA 2011: the posttranslational modification database.Nucleic Acids Res. 2011; 39: D253-D260Crossref PubMed Scopus (297) Google Scholar, 30Rigbolt K.T. Prokhorova T.A. Akimov V. Henningsen J. Johansen P.T. Kratchmarova I. Kassem M. Mann M. Olsen J.V. Blagoev B. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.Sci. Signal. 2011; 4rs3Crossref PubMed Scopus (331) Google Scholar, 31Lundby A. Secher A. Lage K. Nordsborg N.B. Dmytriyev A. Lundby C. Olsen J.V. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues.Nat. Commun. 2012; 3: 876Crossref PubMed Scopus (228) Google Scholar, 32Zhou H. Di Palma S. Preisinger C. Peng M. Polat A.N. Heck A.J. Mohammed S. Toward a comprehensive characterization of a human cancer cell phosphoproteome.J. Proteome Res. 2013; 12: 260-271Crossref PubMed Scopus (248) Google Scholar, 33Bian Y. Song C. Cheng K. Dong M. Wang F. Huang J. Sun D. Wang L. Ye M. Zou H. An enzyme assisted RP-RPLC approach for in-depth analysis of human liver phosphoproteome.J. Proteomics. 2014; 96: 253-262Crossref PubMed Scopus (141) Google Scholar). However, it is currently not known whether Rac activity regulates Myo9b phosphorylation which in turn might regulate motor activity. As shown in the current work, to rescue the morphology and migration phenotypes of Myo9b-deficient cells, local accumulation of Myo9b in protruding lamellipodia was required in addition to Myo9b RhoGAP activity. This implies that global RhoGAP activity by cytosolic Myo9b is not sufficient to compensate for the loss of endogenous Myo9b. It was notable that levels of recombinant Myo9b as low as 20% of the endogenous Myo9b were sufficient to rescue the phenotypes. On the other hand, Myo9b motor mutants at more than twice this expression level were not able to correct the null phenotypes. Cell morphology parameters exhibited values that were somewhere in between those determined for Myo9b-deficient cells that expressed just EGFP or recombinant Myo9b WT. In contrast, cell migration velocity and directionality were not improved by the expression of the Myo9b motor mutants. These results suggest that inhibition of Rho activity globally in the cell by Myo9b slightly modifies cell morphology, but local inhibition of Rho activity by Myo9b at the leading edge is essential for cell migration (Fig. 9). We assume that the two separate missense mutations that are predicted to abrogate motor activity do not affect a potential regulation of the RhoGAP activity of Myo9b. Based on our current results, it is rather the proper subcellular positioning of the RhoGAP domain by the myosin motor that is critical for the regulation of cellular Rho signaling by Myo9b. The spatial control of Rho signaling is critical for cell migration. Initially, it was attributed to the local activity of RhoGEFs that activate Rho at the sides and back of migrating cells (34Wong K. Van Keymeulen A. Bourne H.R. PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells.J. Cell Biol. 2007; 179: 1141-1148Crossref PubMed Scopus (56) Google Scholar). However, it is becoming more and more evident that RhoGAPs