scholarly journals R-Ras Controls Membrane Protrusion and Cell Migration through the Spatial Regulation of Rac and Rho

2005 ◽  
Vol 16 (1) ◽  
pp. 84-96 ◽  
Author(s):  
Michele A. Wozniak ◽  
Lina Kwong ◽  
David Chodniewicz ◽  
Richard L. Klemke ◽  
Patricia J. Keely
Keyword(s):  
Cell Migration ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Membrane Protrusion ◽  
Spatial Regulation ◽  
Entire Cell ◽  
Migratory Cells

Although it is known that the spatial coordination of Rac and Rho activity is essential for cell migration, the molecular mechanisms regulating these GTPases during migration are unknown. We found that the expression of constitutively activated R-Ras (38V) blocked membrane protrusion and random migration. In contrast, expression of dominant negative R-Ras (41A) enhanced migrational persistence and membrane protrusion. Endogenous R-Ras is necessary for cell migration, as cells that were transfected with siRNA for R-Ras did not migrate. Expression of R-Ras (38V) decreased Rac activity and increased Rho activity around the entire cell periphery, whereas expression of dominant negative R-Ras (41A) showed the converse, suggesting that R-Ras can spatially activate Rho and inactivate Rac. Consistent with this role, endogenous R-Ras localized and was preferentially activated at the leading edge of migratory cells in response to adhesion. The effects of R-Ras on cell migration are mediated by PI3-Kinase, as an effector mutant that uncouples PI3-Kinase binding from R-Ras (38V) rescued migration. From these data, we hypothesize that R-Ras plays a key role in cell migration by locally regulating the switch from Rac to Rho activity after membrane protrusion and adhesion.

scholarly journals Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility

2006 ◽  
Vol 176 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Erik Sahai ◽  
Raquel Garcia-Medina ◽  
Jacques Pouysségur ◽  
Emmanuel Vial
Keyword(s):  
Cell Migration ◽  
Tumor Cell ◽  
Rho Gtpases ◽  
Rho Kinase ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Periphery ◽  
Tumor Cell Migration ◽  
Tumor Cell Motility

Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.

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

2008 ◽  
Vol 19 (11) ◽  
pp. 4930-4941 ◽  
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.

scholarly journals βcap73-ARF6 Interactions Modulate Cell Shape and Motility after Injury In Vitro

2003 ◽  
Vol 14 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
Kathleen N. Riley ◽  
Angel E. Maldonado ◽  
Patrice Tellier ◽  
Crislyn D'Souza-Schorey ◽  
Ira M. Herman
Keyword(s):  
Western Blot ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Capping Protein ◽  
Actin Capping Protein

To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, β-actin, or β-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with β-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the β-actin capping protein, βcap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with βcap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and β-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.

scholarly journals Phosphoinositide 3-Kinase C2β Regulates Cytoskeletal Organization and Cell Migration via Rac-dependent Mechanisms

2006 ◽  
Vol 17 (9) ◽  
pp. 3729-3744 ◽  
Author(s):  
Roy M. Katso ◽  
Olivier E. Pardo ◽  
Andrea Palamidessi ◽  
Clemens M. Franz ◽  
Marin Marinov ◽  
...  
Keyword(s):  
Cell Migration ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Human Tumor ◽  
Class Ii ◽  
Dominant Negative ◽  
Class I ◽  
Membrane Ruffling ◽  
And Migration ◽  
Phosphoinositide 3 Kinase

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein–protein and protein–lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2β (PI3KC2β) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2β stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2β reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2β-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2β regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

scholarly journals Curvature sensing steers actin-driven cell migration

2021 ◽  
Author(s):  
Ewa Sitarska ◽  
Silvia Dias Almeida ◽  
Marianne Beckwith ◽  
Julian Stopp ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Membrane Curvature ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Complex Environments ◽  
Time Resolved ◽  
Curvature Sensing ◽  
Membrane Topography

Cell migration is fundamental for the immune response, development, and morphogenesis. For navigation through complex and ever-changing environments, migrating cells require a balance between a stable leading-edge, which is necessary for directional migration, and some unstable features to enable the required dynamic behaviors. The leading edge is often composed of actin-driven protrusions including lamellipodia and ruffles with continuously changing membrane curvature. Whether their membrane topography affects the cell's leading edge and motion persistence in complex environments remains unknown. To study this, we combined a theoretical analysis with machine learning-based segmentation for time-resolved TIRF microscopy, membrane topography analysis from electron microscopy images and microfluidics. We discovered that cell motion persistence and directionality, in both freely moving and environmentally-constrained cells, strongly depend on the curvature-sensing protein Snx33. Specifically, Snx33 promotes leading edge instabilities by locally inhibiting WAVE2- driven actin polymerization in a curvature-dependent manner. Snx33 knockout cells migrate faster and are more persistent during unobstructed migration, but fail when a change in direction is required. Thus, Snx33 is key for steering cell motility in complex environments by facilitating contact inhibition of locomotion and promoting efficient turning. These results identify cell surface topography as an organizing principle at the cell periphery that directs cell migration.

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 Molecular mechanisms of invadopodium formation

2005 ◽  
Vol 168 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hideki Yamaguchi ◽  
Mike Lorenz ◽  
Stephan Kempiak ◽  
Corina Sarmiento ◽  
Salvatore Coniglio ◽  
...  
Keyword(s):  
Rna Interference ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
De Novo ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Matrix Degradation ◽  
Degradation Activity

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

scholarly journals Vimentin organization modulates the formation of lamellipodia

2011 ◽  
Vol 22 (8) ◽  
pp. 1274-1289 ◽  
Author(s):  
Brian T. Helfand ◽  
Melissa G. Mendez ◽  
S. N. Prasanna Murthy ◽  
Dale K. Shumaker ◽  
Boris Grin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Motility ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Entire Cell ◽  
P21 Activated Kinase ◽  
Kinase Phosphorylation ◽  
Negative Mutant

Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.

scholarly journals Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration

2017 ◽  
Vol 28 (16) ◽  
pp. 2159-2169 ◽  
Author(s):  
Max-Hinderk Schuler ◽  
Agnieszka Lewandowska ◽  
Giuseppe Di Caprio ◽  
Wesley Skillern ◽  
Srigokul Upadhyayula ◽  
...  
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Energy Production ◽  
Rho Gtpase ◽  
Leading Edge ◽  
Dorsal Surface ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Intracellular Energy

It has long been postulated, although never directly demonstrated, that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually decreased as more-peripheral sites were approached. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane, which rapidly decreased toward the dorsal surface. We used cells deficient for the mitochondrial Rho-GTPase 1 (Miro1), an essential mediator of microtubule-based mitochondrial motility, to study how changes in mitochondrial positioning affect cytoplasmic energy distribution and cell migration, an energy-expensive process. The mitochondrial network in Miro1−/− MEFs was restricted to the perinuclear area, with few mitochondria present at the cell periphery. This change in mitochondrial distribution dramatically reduced the ratio of ATP to ADP at the cell cortex and disrupted events essential for cell movement, including actin dynamics, lamellipodia protrusion, and membrane ruffling. Cell adhesion status was also affected by changes in mitochondrial positioning; focal adhesion assembly and stability was decreased in Miro1−/−MEFs compared with Miro1+/+ MEFs. Consequently Miro1−/− MEFs migrated slower than control cells during both collective and single-cell migration. These data establish that Miro1-mediated mitochondrial positioning at the leading edge provides localized energy production that promotes cell migration by supporting membrane protrusion and focal adhesion stability.

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