Molecular Regulation of Actin Turnover at the Leading Edge of Migrating Cells

Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells.

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Integrin-mediated Protein Kinase A Activation at the Leading Edge of Migrating Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0564 ◽

2008 ◽

Vol 19 (11) ◽

pp. 4930-4941 ◽

Cited By ~ 59

Author(s):

Chinten J. Lim ◽

Kristin H. Kain ◽

Eugene Tkachenko ◽

Lawrence E. Goldfinger ◽

Edgar Gutierrez ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Protein Kinase A ◽

Leading Edge ◽

Pi3 Kinase ◽

Early Event ◽

Type I ◽

Cell Protrusion ◽

Kinase A ◽

Migrating Cells

cAMP-dependent protein kinase A (PKA) is important in processes requiring localized cell protrusion, such as cell migration and axonal path finding. Here, we used a membrane-targeted PKA biosensor to reveal activation of PKA at the leading edge of migrating cells. Previous studies show that PKA activity promotes protrusion and efficient cell migration. In live migrating cells, membrane-associated PKA activity was highest at the leading edge and required ligation of integrins such as α4β1 or α5β1 and an intact actin cytoskeleton. α4 integrins are type I PKA-specific A-kinase anchoring proteins, and we now find that type I PKA is important for localization of α4β1 integrin-mediated PKA activation at the leading edge. Accumulation of 3′ phosphorylated phosphoinositides [PtdIns(3,4,5)P3] products of phosphatidylinositol 3-kinase (PI3-kinase) is an early event in establishing the directionality of migration; however, polarized PKA activation did not require PI3-kinase activity. Conversely, inhibition of PKA blocked accumulation of a PtdIns(3,4,5)P3-binding protein, the AKT-pleckstrin homology (PH) domain, at the leading edge; hence, PKA is involved in maintaining cell polarity during migration. In sum, we have visualized compartment-specific PKA activation in migrating cells and used it to reveal that adhesion-mediated localized activation of PKA is an early step in directional cell migration.

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Scribble, Lgl1, and myosin II form a complex in vivo to promote directed cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e19-11-0657 ◽

2020 ◽

Vol 31 (20) ◽

pp. 2234-2248

Author(s):

Maha Abedrabbo ◽

Shoshana Ravid

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Myosin Ii ◽

Leading Edge ◽

Directed Cell Migration ◽

Lethal Giant Larvae ◽

And Migration ◽

Migrating Cells

Here we show that Scribble (Scrib), Lethal giant larvae 1 (Lgl1), and myosin II form a complex in vivo and colocalize at the cell leading edge of migrating cells, and this colocalization is interdependent. Scrib and Lgl1 are required for proper cell adhesion, polarity, and migration.

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Cell migration without a lamellipodium

The Journal of Cell Biology ◽

10.1083/jcb.200406063 ◽

2005 ◽

Vol 168 (4) ◽

pp. 619-631 ◽

Cited By ~ 206

Author(s):

Stephanie L. Gupton ◽

Karen L. Anderson ◽

Thomas P. Kole ◽

Robert S. Fischer ◽

Aaron Ponti ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Cells ◽

Leading Edge ◽

Actin Binding ◽

Retrograde Flow ◽

Actin Assembly ◽

Migration Inhibition ◽

Actin Networks ◽

Actin Turnover ◽

High Concentrations

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin–binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle αTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

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Decision letter: The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

10.7554/elife.17440.025 ◽

2016 ◽

Keyword(s):

Focal Adhesions ◽

Leading Edge ◽

Ubiquitin Proteasome System ◽

Ubiquitin Proteasome ◽

Migrating Cells

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Mechanistic of Rac and Cdc42 synchronization at the cell edge by ARHGAP39-dependent signaling nodules and the impact on protrusion dynamics

10.1101/2021.05.13.443687 ◽

2021 ◽

Author(s):

Michael Howell ◽

Violaine Delorme-Walker ◽

Christopher Welch ◽

Ritu Pathak ◽

Klaus M Hahn ◽

...

Keyword(s):

Cell Invasion ◽

Src Kinase ◽

Leading Edge ◽

Gtpase Activating Protein ◽

Invasion And Metastasis ◽

Cell Edge ◽

Motile Cells ◽

Myosin X ◽

The Impact ◽

Migrating Cells

Compartmentalization of GTPase regulators into signaling nodules dictates the GTPase pathways selected. Rac and Cdc42 are synchronized at the cell edge for effective protrusion in motile cells but how their activity is coordinated remains elusive. Here, we discovered that ARHGAP39, a Rac and Cdc42 GTPase-activating protein, sequentially interacts with WAVE and mDia2 to control Rac/lamellipodia and Cdc42/filopodia protrusions, respectively. Mechanistically, ARHGAP39 binds to WAVE and, upon phosphorylation by Src kinase, inactivates Rac to promote Cdc42-induced filopodia formation. With our optimized FRET biosensor, we detected active Cdc42 at the filopodia tips that controls filopodia extension. ARHGAP39 is transported to filopodia tips by Myosin-X where it binds mDia2 and inactivates Cdc42 leading to filopodia retraction. Failure in lamellipodia to filopodia switch by defective ARHGAP39 impairs cell invasion and metastasis. Our study reveals that compartmentalization of ARHGAP39 within Rac/Cdc42 signaling nodules orchestrates the synchronization of lamellipodia/filopodia crosstalk and highlights the intricate regulation of leading edge dynamics in migrating cells.

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The novel RacE-binding protein GflB sharpens Ras activity at the leading edge of migrating cells

Molecular Biology of the Cell ◽

10.1091/mbc.e15-11-0796 ◽

2016 ◽

Vol 27 (10) ◽

pp. 1596-1605 ◽

Cited By ~ 10

Author(s):

Hiroshi Senoo ◽

Huaqing Cai ◽

Yu Wang ◽

Hiromi Sesaki ◽

Miho Iijima

Keyword(s):

Rho Gtpases ◽

Rho Gtpase ◽

Gene Knockout ◽

Binding Protein ◽

Affinity Purification ◽

Leading Edge ◽

The Novel ◽

Gradient Sensing ◽

Signaling Events ◽

Migrating Cells

Directional sensing, a process in which cells convert an external chemical gradient into internal signaling events, is essential in chemotaxis. We previously showed that a Rho GTPase, RacE, regulates gradient sensing in Dictyostelium cells. Here, using affinity purification and mass spectrometry, we identify a novel RacE-binding protein, GflB, which contains a Ras GEF domain and a Rho GAP domain. Using biochemical and gene knockout approaches, we show that GflB balances the activation of Ras and Rho GTPases, which enables cells to precisely orient signaling events toward higher concentrations of chemoattractants. Furthermore, we find that GflB is located at the leading edge of migrating cells, and this localization is regulated by the actin cytoskeleton and phosphatidylserine. Our findings provide a new molecular mechanism that connects directional sensing and morphological polarization.

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Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

The Journal of Cell Biology ◽

10.1083/jcb.201202041 ◽

2012 ◽

Vol 199 (2) ◽

pp. 331-345 ◽

Cited By ~ 50

Author(s):

Shujie Wang ◽

Takashi Watanabe ◽

Kenji Matsuzawa ◽

Akira Katsumi ◽

Mai Kakeno ◽

...

Keyword(s):

Protein Kinase C ◽

Cell Migration ◽

Protein Kinase ◽

Leading Edge ◽

Cell Spreading ◽

Kinase C ◽

Rac1 Activity ◽

Directional Movement ◽

And Migration ◽

Migrating Cells

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

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Regulation of Rho GTPase activity at the leading edge of migrating cells by p190RhoGAP

Small GTPases ◽

10.1080/21541248.2017.1280584 ◽

2017 ◽

Vol 10 (2) ◽

pp. 99-110 ◽

Cited By ~ 5

Author(s):

Aurélien Bidaud-Meynard ◽

Fabien Binamé ◽

Valérie Lagrée ◽

Violaine Moreau

Keyword(s):

Rho Gtpase ◽

Leading Edge ◽

Gtpase Activity ◽

Migrating Cells

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The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation

The Journal of Cell Biology ◽

10.1083/jcb.200703178 ◽

2007 ◽

Vol 179 (2) ◽

pp. 229-238 ◽

Cited By ~ 84

Author(s):

Hiroshi Tokuo ◽

Katsuhide Mabuchi ◽

Mitsuo Ikebe

Keyword(s):

Motor Activity ◽

Motor Function ◽

Leading Edge ◽

Living Cells ◽

Cell Periphery ◽

Actin Reorganization ◽

Cell Biol ◽

Actin Fiber ◽

Myosin X ◽

Migrating Cells

Filopodia are actin-rich fingerlike protrusions found at the leading edge of migrating cells and are believed to play a role in directional sensing. Previous studies have shown that myosin-X (myoX) promotes filopodia formation and that this is mediated through its ability to deliver specific cargoes to the cell periphery (Tokuo, H., and M. Ikebe. 2004. Biochem Biophys. Commun. 319:214–220; Zhang, H., J.S. Berg, Z. Li, Y. Wang, P. Lang, A.D. Sousa, A. Bhaskar, R.E. Cheney, and S. Stromblad. 2004. Nat. Cell Biol. 6:523–531; Bohil, A.B., B.W. Robertson, and R.E. Cheney. 2006. Proc. Natl. Acad. Sci. USA. 103:12411–12416; Zhu, X.J., C.Z. Wang, P.G. Dai, Y. Xie, N.N. Song, Y. Liu, Q.S. Du, L. Mei, Y.Q. Ding, and W.C. Xiong. 2007. Nat. Cell Biol. 9:184–192). In this study, we show that the motor function of myoX and not the cargo function is critical for initiating filopodia formation. Using a dimer-inducing technique, we find that myoX lacking its cargo-binding tail moves laterally at the leading edge of lamellipodia and induces filopodia in living cells. We conclude that the motor function of the two-headed form of myoX is critical for actin reorganization at the leading edge, leading to filopodia formation.

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