Local Myo9b RhoGAP activity regulates cell motility

Abstract

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. 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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 Myo9b with Myo9b mutants GAP or motor activity and the motility of these We that Rac activity is sufficient for Myo9b recruitment to lamellipodial and that local recruitment of Myo9b RhoGAP activity is important for directional cell migration. Myo9b motor activity Myo9b to dynamic actin networks that 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). Myo9b motor activity and in protruding lamellipodia is regulated is not To Rac-induced signaling and actin polymerization be sufficient for the recruitment of Myo9b, we cells with Rac1 Local of protrusive lamellipodia with or a RhoGAP that both at the front of protruding lamellipodia Myo9b localized to the dynamic actin filaments that lamellipodia protrusion as by The recruitment of Myo9b to the leading edge its motor activity. motor mutants that are in nucleotide A. P. of the myosin and actin sites are for C. Full Text PDF PubMed Scopus Google or hydrolysis T. R. K. of the in the of myosin PubMed Scopus Google Scholar, M. S. M.A. D.J. of the and of Mol. Biol. PubMed Scopus Google not recruited to the leading edge of protruding lamellipodia and localized to the protein that Rac-induced lamellipodia leads to the recruitment of Myo9b and that its motor activity is for this HL-60 cells Myo9b and be into macrophages and In to macrophages, they be and To the function of Myo9b in HL-60–derived macrophages, we the Myo9b using shown in two by and a cell by The Myo9b of Myo9b cell by which the of or deletion of In with the the of the Myo9b protein in the cell as shown in Cell that not by as and Myo9b-deficient HL-60 cells into In HL-60 cells Myo9b not altered cell by the of the protein of Myo9b the to the HL-60 that is not by Myo9b we whether Myo9b regulates the morphology of HL-60 we the of cell the the and the of a different Myo9b-deficient a cells of a in a for the Myo9b-deficient cells in cells and a of a not HL-60 cell to Myo9b protein increases during HL-60 of the cell of in and HL-60 in the of and Myo9b-deficient of a are shown for a and a Myo9b-deficient cell of cells of indicated cell that or with for are not by the of Myo9b HL-60 cell HL-60 cells to macrophages in a different cell morphology to whether they Myo9b or not of cells revealed that the Myo9b-deficient cells not a but also are well polarized as indicated by an increased and a of a to cells cell A of as not as a as and as we the migration of HL-60 macrophages on macrophages and with a macrophages the different Myo9b-deficient and of the of migration revealed Myo9b-deficient macrophages the two macrophages the Myo9b-deficient and the Myo9b-deficient but on cells cells of the two To whether the altered morphology, migration and of Myo9b-deficient HL-60 macrophages be by the of we cell of the Myo9b-deficient cell and that or as a to whether the in Myo9b-deficient HL-60 cells are to global or local regulation of Rho activity by we expressed Myo9b that its RhoGAP activity or its motor activity and The of the different the Myo9b-deficient expressed and to of Myo9b in The two different Myo9b to motor activity expressed at the of the other two and In cell the Myo9b-deficient expressed to of the Myo9b level, whereas expressed at of Myo9b of the cell that by revealed that cells of a expressed their at the Myo9b and at the leading edge of protruding whereas the two Myo9b motor and not and the as we the of different Myo9b to the cell morphology of Myo9b-deficient cells to a The of in Myo9b-deficient cells of and cell as shown by an in by the whereas cells that expressed a is that of of the Myo9b in the cells sufficient to the cell In with the that the by Myo9b-deficient cells is a of RhoA the of not cell expressed at the Myo9b The motor that not at the leading edge also not cell of the hydrolysis motor increased cell to that cell be by RhoGAP activity. of the revealed that of Myo9b in the Myo9b but not the to a to that with Myo9b RhoGAP as well as both motor mutants the at all for the of a increased to of cells Myo9b but increased of Myo9b RhoGAP or motor mutants we whether the of different Myo9b in Myo9b-deficient HL-60 macrophages not their morphology but also their migration. The of Myo9b in Myo9b-deficient cells or increased migration and to cell However, of not the and of the migration on the RhoGAP activity of Myo9b. of a and of migration In Myo9b-deficient cells both and and of migration not altered of a Myo9b To for of the motor activity of Myo9b in regulating cell migration, we the migration of cells that of two motor that are to nucleotide and hydrolysis of increased of the cells A and that motor activity is the of the motor not cells that or directional the Myo9b-deficient cells A and that of the functional RhoGAP domain by the motor activity regulates cell migration and Myo9b the GTP hydrolysis of RhoA and it to its inactive GDP-bound J. D. Hall A. C. Bähler M. A of myosin in by rho J. PubMed Scopus Google Scholar, U. J. Bähler M. The myosin is a GTPase-activating protein for rho in role of Biol. Cell. PubMed Scopus (74) Google Scholar). In 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). it not known whether Myo9b regulates cell morphology and migration by Rho GTPase activity in cells or we that Myo9b controls cell morphology and migration by negatively regulating Rho activity locally at sites of actin polymerization as at the leading edge of migrating We have that Myo9b accumulates at the leading edge of protruding lamellipodia with actin filaments that protrusion. for this 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). we that activation of Rac is sufficient to the of Myo9b to extending lamellipodia. Rac activation induces actin and the motor domain of Myo9b with actin filaments or is by the that Myo9b also accumulates at (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). the Myo9b motor could be an to an by a signaling that acts in to actin sites have been in Myo9b H. M. F. R. Z. G. O. M. enables of the the cell Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. S.A. S.J. A of Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J.E. T. R. S.A. J. W. A of protein and 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, F. J. M. the PubMed Scopus Google Scholar, J. I. M. M. B. of the and of cell PubMed Scopus Google Scholar, A. A. K. A. C. of protein sites different and 2012; PubMed Scopus Google Scholar, H. S. C. M. S. a of a cell 2013; PubMed Scopus Google Scholar, Y. Song C. K. M. Wang F. J. D. Wang L. M. H. approach for of 2014; PubMed Scopus Google Scholar). However, it is not known whether Rac activity regulates Myo9b which in regulate motor activity. shown in the to the morphology and migration of Myo9b-deficient local of Myo9b in protruding lamellipodia in to Myo9b RhoGAP activity. that global RhoGAP activity by Myo9b is not sufficient to for the of Myo9b. that of recombinant Myo9b as as of the Myo9b sufficient to the the other Myo9b motor mutants at this not to the Cell morphology that in for Myo9b-deficient cells that expressed or recombinant Myo9b In cell migration and not by the of the Myo9b motor that inhibition of Rho activity in the cell by Myo9b cell morphology, but local inhibition of Rho activity by Myo9b at the leading edge is for cell migration We assume that the two that are to motor activity not a regulation of the RhoGAP activity of Myo9b. on it is the subcellular of the RhoGAP domain by the myosin motor that is for the regulation of cellular Rho signaling by Myo9b. The of Rho signaling is for cell migration. it to the local activity of that Rho at the sides and back of migrating cells K. Keymeulen A. Bourne H.R. and myosin II RhoA activity to the back of cells.J. Cell Biol. 2007; PubMed Scopus Google Scholar). However, it is and that are in local regulation of Rho signaling as shown for Myo9b. Myo9b a Rac and Rho signaling by Rho at sites of Rac activity. is to reinforce extension and prevent it that protrusive locally the activation of RhoA K. Pertz O. Hahn K. Bourne H. spatiotemporal dynamics of RhoA activity a self-organizing Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). cells different and of have been in the regulation of cell migration C.D. Rho GTPase signaling in cell migration and Cell Biol. 2018; PubMed Scopus Google Scholar). In the it be important to Rho signaling at the cell front is coordinated and by these different RhoGAP In we that Rac activation leads to actin polymerization and local inhibition of Rho activity by the recruitment of the RhoGAP Myo9b. Rac and Rho that is by Myo9b cell migration. The for D. A. I. B. Hahn K.M. A Rac controls the motility of cells.Nature. 2009; 461: PubMed Scopus Google The for J. A.H. K. D. M. F. D. Z. M. R. a to PubMed Scopus Google by R. by for in using the The and by in the (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 the for with that of the for Myo9b, and into the cells in with class HL-60 cells a of Schmidt in with and They a to by to cells a of toward macrophages in by the of to HL-60 for to cells to cells at in a with cells a in of and as a to the In of of of and of in of with of and for at of to this and to the which been with and with of of the replaced by and cells for the to with of Myo9b, and for to two into the Myo9b that two each these the for the and a The a of B. for and is a of the the L. D. S. R. Wu X. W. F. using 2013; PubMed Scopus Google Scholar, S. A. S. Y. F. by for enhanced 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). of the the of into as S. A. S. Y. F. by for enhanced 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). Both using and a II to the to the cells into using a with the of M. for and by and protein by using Myo9b E. A. Bähler M. is a myosin expressed in Cell Sci. PubMed Google Scholar). generated as J. M. S. J. K. T. B. E. E. H. actin and dynamics regulate and cell migration during PubMed Scopus (59) Google Scholar). HL-60 cells in or of and in of or of Myo9b to each well to a of The for at cells carefully to of in The the replaced by the cells and them in the cells for at using a cells and by cell Rac1 with cells on an with an and with and an an a and a cells using the to activation of A of by a with at a with a of at the cell edge to the protruding of the in cells with at the of the The toward Rac1 and of the proteins with the with an of the cell for with the function of the The to of the at To the of Rac1 cells of these with of their during and for Rac1 using of the generated using the by J. and A. 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