scholarly journals Cell migration without a lamellipodium

2005 ◽  
Vol 168 (4) ◽  
pp. 619-631 ◽  
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.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals A pathway of neuregulin-induced activation of cofilin-phosphatase Slingshot and cofilin in lamellipodia

2004 ◽  
Vol 165 (4) ◽  
pp. 465-471 ◽  
Author(s):  
Kyoko Nagata-Ohashi ◽  
Yusaku Ohta ◽  
Kazumichi Goto ◽  
Shuhei Chiba ◽  
Reiko Mori ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Filaments ◽  
Leading Edge ◽  
Actin Assembly ◽  
Filament Dynamics ◽  
Signaling Mechanisms ◽  
Actin Turnover ◽  
Per Se ◽  
Activity Dependent ◽  
Stimulation Of

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases ∼10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1β induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin–rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1β and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.

scholarly journals Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

2013 ◽  
Vol 202 (1) ◽  
pp. 163-177 ◽  
Author(s):  
Ingo Thievessen ◽  
Peter M. Thompson ◽  
Sylvain Berlemont ◽  
Karen M. Plevock ◽  
Sergey V. Plotnikov ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Leading Edge ◽  
Flow Speed ◽  
Actin Binding ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Primary Fibroblasts

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.

Nanotopography-Modulated Epithelial Cell Collective Migration

2021 ◽  
Vol 17 (6) ◽  
pp. 1079-1087
Author(s):  
Zaozao Chen ◽  
Qiwei Li ◽  
Shihui Xu ◽  
Jun Ouyang ◽  
Hongmei Wei
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Epithelial Cells ◽  
Leading Edge ◽  
Cell Types ◽  
Protein Kinase Activity ◽  
Migration Behavior ◽  
Collective Migration ◽  
Epithelial Migration ◽  
Activity Dependent

Matrix nanotopography plays an essential role in regulating cell behaviors including cell proliferation, differentiation, and migration. While studies on isolated single cell migration along the nanostructural orientation have been reported for various cell types, there remains a lack of understanding of how nanotopography regulates the behavior of collectively migrating cells during processes such as epithelial wound healing. We demonstrated that collective migration of epithelial cells was promoted on nanogratings perpendicular to, but not on those parallel to, the wound-healing axis. We further discovered that nanograting-modulated epithelial migration was dominated by the adhesion turnover process, which was Rho-associated protein kinase activity-dependent, and the lamellipodia protrusion at the cell leading edge, which was Rac1-GTPase activity-dependent. This work provides explanations to the distinct migration behavior of epithelial cells on nanogratings, and indicates that the effect of nanotopographic modulations on cell migration is cell-type dependent and involves complex mechanisms

scholarly journals Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

1997 ◽  
Vol 139 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Clare M. Waterman-Storer ◽  
E.D. Salmon
Keyword(s):  
Epithelial Cells ◽  
Dynamic Instability ◽  
Leading Edge ◽  
Unknown Origin ◽  
Time Lapse ◽  
Lung Epithelial Cells ◽  
Retrograde Flow ◽  
Microtubule Dynamic ◽  
Lung Epithelial ◽  
Time Lapse Imaging

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 μm/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione–2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that ∼80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

scholarly journals Shootin1–cortactin interaction mediates signal–force transduction for axon outgrowth

2015 ◽  
Vol 210 (4) ◽  
pp. 663-676 ◽  
Author(s):  
Yusuke Kubo ◽  
Kentarou Baba ◽  
Michinori Toriyama ◽  
Takunori Minegishi ◽  
Tadao Sugiura ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Leading Edge ◽  
Growth Cones ◽  
Axon Outgrowth ◽  
Actin Binding ◽  
Retrograde Flow ◽  
Environmental Chemical ◽  
Mechanical Coupling ◽  
Cell Adhesions ◽  
Force Transduction

Motile cells transduce environmental chemical signals into mechanical forces to achieve properly controlled migration. This signal–force transduction is thought to require regulated mechanical coupling between actin filaments (F-actins), which undergo retrograde flow at the cellular leading edge, and cell adhesions via linker “clutch” molecules. However, the molecular machinery mediating this regulatory coupling remains unclear. Here we show that the F-actin binding molecule cortactin directly interacts with a clutch molecule, shootin1, in axonal growth cones, thereby mediating the linkage between F-actin retrograde flow and cell adhesions through L1-CAM. Shootin1–cortactin interaction was enhanced by shootin1 phosphorylation by Pak1, which is activated by the axonal chemoattractant netrin-1. We provide evidence that shootin1–cortactin interaction participates in netrin-1–induced F-actin adhesion coupling and in the promotion of traction forces for axon outgrowth. Under cell signaling, this regulatory F-actin adhesion coupling in growth cones cooperates with actin polymerization for efficient cellular motility.

scholarly journals MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

2020 ◽  
Author(s):  
Chiara Galloni ◽  
Davide Carra ◽  
Jasmine V. G. Abella ◽  
Svend Kjær ◽  
Pavithra Singaravelu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Actin Filament ◽  
Actin Assembly ◽  
Actin Network ◽  
Network Stability ◽  
Actin Networks ◽  
Cellular Processes ◽  
Actin Turnover ◽  
Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

scholarly journals Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

2021 ◽  
Author(s):  
Frieda Kage ◽  
Hermann Doering ◽  
Magdalena Mietkowska ◽  
Matthias Schaks ◽  
Franziska Gruener ◽  
...  
Keyword(s):  
Cell Migration ◽  
Growth Factor ◽  
Genome Editing ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Actin Remodeling ◽  
Actin Networks ◽  
Rac Gtpases ◽  
Signaling Axis ◽  
Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

scholarly journals Tracking Retrograde Flow in Keratocytes: News from the Front

2005 ◽  
Vol 16 (3) ◽  
pp. 1223-1231 ◽  
Author(s):  
Pascal Vallotton ◽  
Gaudenz Danuser ◽  
Sophie Bohnet ◽  
Jean-Jacques Meister ◽  
Alexander B. Verkhovsky
Keyword(s):  
Actin Polymerization ◽  
Leading Edge ◽  
Membrane Resistance ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Actin Network ◽  
Freely Moving ◽  
Contractile Forces ◽  
Cell Motion ◽  
Shape Changes

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1–3 μm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

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