Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces

Parker, Kevin Kit; Brock, Amy; Brangwynne, Cliff; Mannix, Robert; Wang, Ning; Ostuni, Emanuele; Geisse, Nicholas A.; Adams, Josephine C.; Whitesides, George M.; Ingber, Donald E.

doi:10.1096/fj.02-0038com

Cited by 424 publications

(432 citation statements)

References 56 publications

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“…3a and Supplementary Movies 1 and 2). In cells plated on square adhesive patterns, central actin stress fibres were observed to preferentially extent diagonally, in line with previous studies on fibroblast cells 34 . It is noteworthy that this result is consistent with the diagonal orientations of the nucleus that we observed on square-shaped cells (Fig.…”

Section: Resultssupporting

confidence: 91%

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

2012

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Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. on the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

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Section: Resultssupporting

confidence: 91%

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

2012

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“…By extension to living cells, this work emphasizes the importance of the spatial and temporal organization of the nucleation areas, giving rise to specific actin-network architectures and hence controlling the location of force production. Although the spatio-temporal regulation of actin growth is known to influence cell shape 27,28 , our work has revealed, in quantitative terms, that reciprocally physical boundaries, within or around the cell, control actin cytoskeleton architectures.…”

Section: Nature Materialsmentioning

confidence: 85%

Nucleation geometry governs ordered actin networks structures

et al. 2010

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Actin filaments constitute one of the main components of cell cytoskeleton. Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] . However, the question of how geometrical boundaries, such as those encountered in cells, affect the dynamic formation of highly ordered actin structures remains poorly studied 14,19 . Here we demonstrate that the nucleation geometry in itself can be the principal determinant of actin-network architecture. We developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays. Shape, orientation and distance between nucleation regions control filament orientation and length, filament-filament interactions and filopodium-like bundle formation. Modelling of filament growth and interactions demonstrates that basic mechanical and probabilistic laws govern actin assembly in higher-order structures.In cells, actin nucleation occurs at various locations at the plasma membrane, and bundles of parallel actin filaments are initiated at focal adhesion sites 1 or result from the rearrangement of the dynamic branched actin network of the lamellipodium 6 . Here, we modulate the positioning of nucleation sites at scales corresponding to cellular dimensions. As a first step and to precisely regulate the position of actin nucleation sites in vitro, we used a recently developed ultraviolet-based micropatterning approach 20 to create a template for the localization of the nucleation promoting factor pWA (Fig. 1a). pWA comprises the C-terminal domains from the WASP/Scar proteins, a ubiquitous family of proteins that initiate actin polymerization on a pre-existing actin filament in the presence of the Arp2/3 complex and an actin monomer [21][22][23] . A small volume of solution made of a minimal set of purified proteins ensuring actin polymerization 8,10 was placed between the pWA-coated micropatterned slide and a glass support. Functionalized micropatterns specifically initiate actin filament nucleation on their surface and promote two-dimensional growth of actin filaments (Fig. 1b). Real-time visualization of actin-filament nucleation and growth highlighted the autocatalytic process of network formation (see Supplementary Fig. S1). These networks consist of filaments growing from the pWA-coated regions, with their fast-growing, barbed end oriented outwards ( Fig. 1c and Supplementary Fig. S2 and Videos S1-S3). In agreement with actin-filament growth on glass rods 15 , as the nucleation waves propagate, dense and interconnected

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“…Tractional forces exerted by integrin-based focal adhesions on the local ECM environment are important for the development of lamellipodial protrusions (Wang et al, 2001;Parker et al, 2002). At the molecular level, substratum adhesion is required for coupling of Rac to its effector p21-activated kinase, and, in cells lacking the focal adhesion protein vinculin, constitutively active Rac is not sufficient to rescue lamellipodia formation (del Pozo et al, 2000;Goldmann and Ingber, 2002).…”

Section: Discussionmentioning

confidence: 99%

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

Hashimoto

Parsons

Adams³

2007

MBoC

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127

169

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Recurrence of carcinomas due to cells that migrate away from the primary tumor is a major problem in cancer treatment. Immunohistochemical analyses of human carcinomas have consistently correlated up-regulation of the actin-bundling protein fascin with a clinically aggressive phenotype and poor prognosis. To understand the functional and mechanistic contributions of fascin, we undertook inducible short hairpin RNA (shRNA) knockdown of fascin in human colon carcinoma cells derived from an aggressive primary tumor. Fascin-depletion led to decreased numbers of filopodia and altered morphology of cell protrusions, decreased Rac-dependent migration on laminin, decreased turnover of focal adhesions, and, in vivo, decreased xenograft tumor development and metastasis. cDNA rescue of fascin shRNAknockdown cells with wild-type green fluorescent protein-fascin or fascins mutated at the protein kinase C (PKC) phosphorylation site revealed that both the actin-bundling and active PKC-binding activities of fascin are required for the organization of filopodial protrusions, Rac-dependent migration, and tumor metastasis. Thus, fascin contributes to carcinoma migration and metastasis through dual pathways that impact on multiple subcellular structures needed for cell migration.

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Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces

Cited by 424 publications

References 56 publications

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Nucleation geometry governs ordered actin networks structures

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

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