scholarly journals Spatial Regulation of MCAK Promotes Cell Polarization and Focal Adhesion Turnover to Drive Robust Cell Migration

2020 ◽  
Author(s):  
Hailing Zong ◽  
Mark Hazelbaker ◽  
Christina Moe ◽  
Stephanie C. Ems-McClung ◽  
Ke Hu ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Polarization ◽  
Asymmetric Distribution ◽  
Membrane Ruffling ◽  
Spatial Regulation ◽  
Directional Movement ◽  
Parallel Pathway ◽  
Focal Adhesion Turnover

AbstractThe asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These defects are due in part to the loss of the spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model that places MCAK at a key nexus of a feedback loop, in which polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contributes to cell polarity and directional migration.

scholarly journals Spatial Regulation of MCAK Promotes Cell Polarization and Focal Adhesion Turnover to Drive Robust Cell Migration

2021 ◽  
pp. mbc.E20-05-0301
Author(s):  
Hailing Zong ◽  
Mark Hazelbaker ◽  
Christina Moe ◽  
Stephanie C. Ems-McClung ◽  
Ke Hu ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Polarization ◽  
Asymmetric Distribution ◽  
Membrane Ruffling ◽  
Spatial Regulation ◽  
Directional Movement ◽  
Focal Adhesion Turnover

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK, in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared to the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning and focal adhesion dynamics that all help facilitate robust directional migration. [Media: see text] [Media: see text]

scholarly journals Role of P120 Ras-Gap in Directed Cell Movement

2000 ◽  
Vol 149 (2) ◽  
pp. 457-470 ◽  
Author(s):  
Sarang V. Kulkarni ◽  
Gerald Gish ◽  
Peter van der Geer ◽  
Mark Henkemeyer ◽  
Tony Pawson
Keyword(s):  
Cell Polarity ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Directional Movement ◽  
Complete Cell ◽  
And Migration

We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP–deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility.

scholarly journals Microtubule acetylation but not detyrosination promotes focal adhesion dynamics and cell migration

2018 ◽  
Author(s):  
Bertille Bance ◽  
Shailaja Seetharaman ◽  
Cécile Leduc ◽  
Batiste Boëda ◽  
Sandrine Etienne-Manneville
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Post Translational Modifications ◽  
Tubulin Acetylation ◽  
Focal Adhesion Turnover ◽  
Regulatory Functions ◽  
Insight Into

AbstractMicrotubules play a crucial role in mesenchymal migration by controlling cell polarity and the turnover of cell adhesive structures on the extracellular matrix. The polarized functions of microtubules imply that microtubules are locally regulated. Here, we investigated the regulation and role of two major tubulin post-translational modifications, acetylation and detyrosination, which have been associated with stable microtubules. Using primary astrocytes in a wound healing assay, we show that these tubulin modifications are independently regulated during cell polarization and differently affect cell migration. In contrast to microtubule detyrosination, αTAT1-mediated microtubule acetylation increases in the vicinity of focal adhesions and promotes cell migration. We further demonstrate that αTAT1 increases focal adhesion turnover by promoting Rab6-positive vesicle fusion at focal adhesions. Our results highlight the specificity of microtubule post-translational modifications and bring new insight into the regulatory functions of tubulin acetylation.

scholarly journals EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 388
Author(s):  
Brice Chanez ◽  
Kevin Ostacolo ◽  
Ali Badache ◽  
Sylvie Thuault
Keyword(s):  
Breast Cancer ◽  
Cancer Cell ◽  
Cell Invasion ◽  
Focal Adhesions ◽  
Matrix Degradation ◽  
Cancer Cell Invasion ◽  
Wild Type ◽  
Spatial Regulation ◽  
Invadopodia Formation

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

scholarly journals Tropomyosin Isoform Expression Regulates the Transition of Adhesions To Determine Cell Speed and Direction

2009 ◽  
Vol 29 (6) ◽  
pp. 1506-1514 ◽  
Author(s):  
Cuc T. T. Bach ◽  
Sarah Creed ◽  
Jessie Zhong ◽  
Maha Mahmassani ◽  
Galina Schevzov ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Actin Filaments ◽  
Feedback Loop ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Mesenchymal Cell ◽  
Critical Determinant ◽  
Focal Complex

ABSTRACT The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.

scholarly journals Daydreamer, a Ras effector and GSK-3 substrate, is important for directional sensing and cell motility

2013 ◽  
Vol 24 (2) ◽  
pp. 100-114 ◽  
Author(s):  
Verena Kölsch ◽  
Zhouxin Shen ◽  
Susan Lee ◽  
Katarzyna Plak ◽  
Pouya Lotfi ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Leading Edge ◽  
Adaptor Proteins ◽  
Cell Polarization ◽  
Glycogen Synthase Kinase 3 ◽  
Dynamic Regulation ◽  
Multiple Substrates ◽  
Directional Movement ◽  
Phosphoinositide 3 Kinase ◽  
Kinetics Of

How independent signaling pathways are integrated to holistically control a biological process is not well understood. We have identified Daydreamer (DydA), a new member of the Mig10/RIAM/lamellipodin (MRL) family of adaptor proteins that localizes to the leading edge of the cell. DydA is a putative Ras effector that is required for cell polarization and directional movement during chemotaxis. dydA− cells exhibit elevated F-actin and assembled myosin II (MyoII), increased and extended phosphoinositide-3-kinase (PI3K) activity, and extended phosphorylation of the activation loop of PKB and PKBR1, suggesting that DydA is involved in the negative regulation of these pathways. DydA is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is required for some, but not all, of DydA's functions, including the proper regulation of PKB and PKBR1 and MyoII assembly. gskA− cells exhibit very strong chemotactic phenotypes, as previously described, but exhibit an increased rate of random motility. gskA− cells have a reduced MyoII response and a reduced level of phosphatidylinositol (3,4,5)-triphosphate production, but a highly extended recruitment of PI3K to the plasma membrane and highly extended kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3 function is essential for chemotaxis, regulating multiple substrates, and that one of these effectors, DydA, plays a key function in the dynamic regulation of chemotaxis.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470 ◽  
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

Abstract This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

scholarly journals A Role for P21-Activated Kinase in Endothelial Cell Migration

1999 ◽  
Vol 147 (4) ◽  
pp. 831-844 ◽  
Author(s):  
William B. Kiosses ◽  
R. Hugh Daniels ◽  
Carol Otey ◽  
Gary M. Bokoch ◽  
Martin Alexander Schwartz
Keyword(s):  
Cell Migration ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Dominant Negative ◽  
Endothelial Cell Migration ◽  
Stress Fibers ◽  
Membrane Ruffling ◽  
Cell Contractility ◽  
P21 Activated Kinase

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.

scholarly journals Phospholipase C Regulation of Phosphatidylinositol 3,4,5-trisphosphate-mediated Chemotaxis

2007 ◽  
Vol 18 (12) ◽  
pp. 4772-4779 ◽  
Author(s):  
Arjan Kortholt ◽  
Jason S. King ◽  
Ineke Keizer-Gunnink ◽  
Adrian J. Harwood ◽  
Peter J.M. Van Haastert
Keyword(s):  
Phospholipase C ◽  
Leading Edge ◽  
Cell Polarization ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatase And Tensin Homolog ◽  
Pi3k Inhibitor Ly294002 ◽  
Tensin Homolog ◽  
Camp Stimulation

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P3 on the membrane nearest the polarizing signal and PI(3,4,5)P3 dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P2, could have important consequences for PI(3,4,5)P3 localization. We investigate the role of PLC in PI(3,4,5)P3-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P3 after cAMP stimulation, as monitored by the PI(3,4,5)P3-specific pleckstrin homology (PH)-domain of CRAC (PHCRACGFP). In contrast, PLC overexpression elevates PI(3,4,5)P3 and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P3 gradient.

Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2462-2470
Author(s):  
Stéphanie Seveau ◽  
Hansuli Keller ◽  
Frederick R. Maxfield ◽  
Friedrich Piller ◽  
Lise Halbwachs-Mecarelli
Keyword(s):  
Confocal Microscopy ◽  
Leading Edge ◽  
Domain Analysis ◽  
Cell Polarization ◽  
Actin Binding ◽  
Cross Linking ◽  
Chemotactic Peptide ◽  
Membrane Molecule ◽  
Stimulation Of

This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.

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