Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

2019 ◽  
Author(s):  
Kristen Skruber ◽  
Peyton Warp ◽  
Rachael Shklyarov ◽  
James D. Thomas ◽  
Maurice Swanson ◽  
...  
Keyword(s):  
Leading Edge ◽  
Actin Network

Actin network organization at the leading edge of crawling cells

2006 ◽  
Vol 39 ◽  
pp. S240
Author(s):  
S. Sun ◽  
D. Wirtz ◽  
E. Atilgan
Keyword(s):  
Leading Edge ◽  
Network Organization ◽  
Actin Network

scholarly journals WAVE1 and WAVE2 have distinct and overlapping roles in controlling actin assembly at the leading edge

2020 ◽  
Vol 31 (20) ◽  
pp. 2168-2178
Author(s):  
Qing Tang ◽  
Matthias Schaks ◽  
Neha Koundinya ◽  
Changsong Yang ◽  
Luther W. Pollard ◽  
...  
Keyword(s):  
Leading Edge ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Nucleation ◽  
Lamellipodia Formation ◽  
Network Extension

This study shows that WAVE1 and WAVE2 have redundant roles as actin nucleation-promoting factors (NPFs) in promoting lamellipodia formation, but also unique and nonoverlapping roles in controlling the rate of actin network extension, with WAVE2 promoting and WAVE1 restricting growth.

scholarly journals Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

2016 ◽  
Vol 113 (42) ◽  
pp. E6352-E6361 ◽  
Author(s):  
Shalin B. Mehta ◽  
Molly McQuilken ◽  
Patrick J. La Riviere ◽  
Patricia Occhipinti ◽  
Amitabh Verma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Leading Edge ◽  
Living Cells ◽  
Molecular Assembly ◽  
Higher Order ◽  
Live Cells ◽  
Actin Network ◽  
Position And Orientation ◽  
Orientation Of Molecules

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.

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 Memo–RhoA–mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells

2008 ◽  
Vol 183 (3) ◽  
pp. 401-408 ◽  
Author(s):  
Kossay Zaoui ◽  
Stéphane Honoré ◽  
Daniel Isnardon ◽  
Diane Braguer ◽  
Ali Badache
Keyword(s):  
Cell Migration ◽  
Leading Edge ◽  
Site Formation ◽  
Actin Assembly ◽  
Actin Network ◽  
Adhesion Sites ◽  
Cell Front ◽  
Guanosine Triphosphatase ◽  
Adhesion Site ◽  
Migrating Cells

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.

scholarly journals Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

2013 ◽  
Vol 200 (5) ◽  
pp. 619-633 ◽  
Author(s):  
Elena Ingerman ◽  
Jennifer Ying Hsiao ◽  
R. Dyche Mullins
Keyword(s):  
Atp Hydrolysis ◽  
Leading Edge ◽  
S2 Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Dendritic Networks ◽  
A Cell

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

scholarly journals Discrete mechanical model of lamellipodial actin network implements molecular clutch mechanism and generates arcs and microspikes

2021 ◽  
Vol 17 (10) ◽  
pp. e1009506
Author(s):  
David M. Rutkowski ◽  
Dimitrios Vavylonis
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Flow Speed ◽  
Viscous Drag ◽  
Stress Profile ◽  
Retrograde Flow ◽  
Actin Network ◽  
Clutch Mechanism

Mechanical forces, actin filament turnover, and adhesion to the extracellular environment regulate lamellipodial protrusions. Computational and mathematical models at the continuum level have been used to investigate the molecular clutch mechanism, calculating the stress profile through the lamellipodium and around focal adhesions. However, the forces and deformations of individual actin filaments have not been considered while interactions between actin networks and actin bundles is not easily accounted with such methods. We develop a filament-level model of a lamellipodial actin network undergoing retrograde flow using 3D Brownian dynamics. Retrograde flow is promoted in simulations by pushing forces from the leading edge (due to actin polymerization), pulling forces (due to molecular motors), and opposed by viscous drag in cytoplasm and focal adhesions. Simulated networks have densities similar to measurements in prior electron micrographs. Connectivity between individual actin segments is maintained by permanent and dynamic crosslinkers. Remodeling of the network occurs via the addition of single actin filaments near the leading edge and via filament bond severing. We investigated how several parameters affect the stress distribution, network deformation and retrograde flow speed. The model captures the decrease in retrograde flow upon increase of focal adhesion strength. The stress profile changes from compression to extension across the leading edge, with regions of filament bending around focal adhesions. The model reproduces the observed reduction in retrograde flow speed upon exposure to cytochalasin D, which halts actin polymerization. Changes in crosslinker concentration and dynamics, as well as in the orientation pattern of newly added filaments demonstrate the model’s ability to generate bundles of filaments perpendicular (actin arcs) or parallel (microspikes) to the protruding direction.

scholarly journals Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia

1999 ◽  
Vol 145 (5) ◽  
pp. 1009-1026 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Gary G. Borisy
Keyword(s):  
Actin Filaments ◽  
Leading Edge ◽  
Actin Network ◽  
Actin Depolymerizing Factor ◽  
Nucleation Model ◽  
Branch Formation ◽  
Cell Front ◽  
Dendritic Organization ◽  
Pointed End

The leading edge (∼1 μm) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 μm of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181–6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

scholarly journals Leading edge stability in motile cells is an emergent property of branched actin network growth

2020 ◽  
Author(s):  
Rikki M. Garner ◽  
Julie A. Theriot
Keyword(s):  
High Speed ◽  
Noise Suppression ◽  
Animal Cell ◽  
Leading Edge ◽  
Lateral Spreading ◽  
Actin Network ◽  
Biological Noise ◽  
Network Growth ◽  
Motile Cells ◽  
Branch Formation

AbstractAnimal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed, high-resolution imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale dynamic shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that is able to reproduce this stable relaxation behavior. Remarkably, we find that lamellipodial stability naturally emerges from the interplay between branched actin network growth and leading edge shape – with no additional feedback required – based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ∼70-80º branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals.One sentence summaryAn experimental and computational investigation of fluctuation dynamics at the leading edge of motile cells demonstrates that the specific angular geometry of Arp2/3-mediated actin network branch formation lies at the core of a successful biological noise-suppression strategy.

scholarly journals The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration

2012 ◽  
Vol 197 (2) ◽  
pp. 239-251 ◽  
Author(s):  
Praveen Suraneni ◽  
Boris Rubinstein ◽  
Jay R. Unruh ◽  
Michael Durnin ◽  
Dorit Hanein ◽  
...  
Keyword(s):  
Cell Migration ◽  
Critical Role ◽  
Embryonic Stem ◽  
Leading Edge ◽  
Fibroblast Cell ◽  
Fibroblast Cells ◽  
Actin Network ◽  
Directional Migration ◽  
Fibroblast Migration ◽  
Motile Cells

The Arp2/3 complex nucleates the formation of the dendritic actin network at the leading edge of motile cells, but it is still unclear if the Arp2/3 complex plays a critical role in lamellipodia protrusion and cell motility. Here, we differentiated motile fibroblast cells from isogenic mouse embryonic stem cells with or without disruption of the ARPC3 gene, which encodes the p21 subunit of the Arp2/3 complex. ARPC3−/− fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions, with formin proteins (mDia1 and mDia2) concentrated near their tips. The speed of cell migration, as well as the rates of leading edge protrusion and retraction, were comparable between genotypes; however, ARPC3−/− cells exhibited a strong defect in persistent directional migration. This deficiency correlated with a lack of coordination of the protrusive activities at the leading edge of ARPC3−/− fibroblasts. These results provide insights into the Arp2/3 complex’s critical role in lamellipodia extension and directional fibroblast migration.

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