scholarly journals Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

2016 ◽  
Vol 27 (9) ◽  
pp. 1431-1441 ◽  
Author(s):  
Aniqua Rahman ◽  
Shawn P. Carey ◽  
Casey M. Kraning-Rush ◽  
Zachary E. Goldblatt ◽  
Francois Bordeleau ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Focal Adhesions ◽  
Three Dimensions ◽  
Collagen Matrices ◽  
Migration Direction ◽  
Unidirectional Motion

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.

scholarly journals The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration

2012 ◽  
Vol 23 (4) ◽  
pp. 591-601 ◽  
Author(s):  
Inbal Dahan ◽  
Ahuv Yearim ◽  
Yarin Touboul ◽  
Shoshana Ravid
Keyword(s):  
Tumor Suppressor ◽  
Cell Polarity ◽  
Focal Adhesion ◽  
Cellular Localization ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Membrane Dynamics ◽  
Migrating Cells

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical–basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.

scholarly journals Epithelial Cell Chirality Revealed by Three-Dimensional Spontaneous Rotation

2018 ◽  
Vol 115 (48) ◽  
pp. 12188-12193 ◽  
Author(s):  
Amanda S. Chin ◽  
Kathryn E. Worley ◽  
Poulomi Ray ◽  
Gurleen Kaur ◽  
Jie Fan ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Cell Polarity ◽  
Three Dimensional ◽  
Intrinsic Property ◽  
Cell Aggregates ◽  
Mechanistic Studies ◽  
Directional Bias ◽  
Self Organized ◽  
Dependent Cell

Our understanding of the left–right (LR) asymmetry of embryonic development, in particular the contribution of intrinsic handedness of the cell or cell chirality, is limited due to the confounding systematic and environmental factors during morphogenesis and a ack of physiologically relevant in vitro 3D platforms. Here we report an efficient two-layered biomaterial platform for determining the chirality of individual cells, cell aggregates, and self-organized hollow epithelial spheroids. This bioengineered niche provides a uniform defined axis allowing for cells to rotate spontaneously with a directional bias toward either clockwise or counterclockwise directions. Mechanistic studies reveal an actin-dependent, cell-intrinsic property of 3D chirality that can be mediated by actin cross-linking via α-actinin-1. Our findings suggest that the gradient of extracellular matrix is an important biophysicochemical cue influencing cell polarity and chirality. Engineered biomaterial systems can serve as an effective platform for studying developmental asymmetry and screening for environmental factors causing birth defects.

scholarly journals Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lillian K Fritz-Laylin ◽  
Megan Riel-Mehan ◽  
Bi-Chang Chen ◽  
Samuel J Lord ◽  
Thomas D Goddard ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Time Lapse ◽  
Three Dimensions ◽  
Cortical Actin ◽  
Linear Arrangement ◽  
Collagen Matrices ◽  
Flat Surfaces ◽  
Actin Networks ◽  
Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

1999 ◽  
Vol 112 (24) ◽  
pp. 4589-4599 ◽  
Author(s):  
F. Li ◽  
Y. Zhang ◽  
C. Wu
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Focal Adhesions ◽  
Subcellular Compartment ◽  
Cell Matrix ◽  
Integrin Binding Site ◽  
Integrin Linked Kinase ◽  
Cell Cell

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Alisson C Cardoso ◽  
Ana H Pereira ◽  
Andre L Ambrosio ◽  
Silvio R Consonni ◽  
Sandra M Dias ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mechanical Stress ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Structural Information ◽  
Mechanistic Model ◽  
Focal Adhesions ◽  
Saxs Data ◽  
X Ray ◽  
Gene Assay

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

scholarly journals Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

2000 ◽  
Vol 348 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Madeleine TOUTANT ◽  
Jeanne-Marie STUDLER ◽  
Ferran BURGAYA ◽  
Alicia COSTA ◽  
Pascal EZAN ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Critical Residue ◽  
Green Fluorescent ◽  
And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

Local, Three-Dimensional Strain Measurements Within Largely Deformed Extracellular Matrix Constructs

2004 ◽  
Vol 126 (6) ◽  
pp. 699-708 ◽  
Author(s):  
Blayne A. Roeder ◽  
Klod Kokini ◽  
J. Paul Robinson ◽  
Sherry L. Voytik-Harbin
Keyword(s):  
Extracellular Matrix ◽  
Local Level ◽  
Three Dimensional ◽  
Collagen Matrices ◽  
Mechanical Loads ◽  
Strain Measurements ◽  
3D Collagen ◽  
Vitro Model

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However, the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. “Self-assembled” collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both 1D and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.

scholarly journals Restructuring of Focal Adhesion Plaques by Pi 3-Kinase

2000 ◽  
Vol 150 (3) ◽  
pp. 627-642 ◽  
Author(s):  
Jeffrey A. Greenwood ◽  
Anne B. Theibert ◽  
Glenn D. Prestwich ◽  
Joanne E. Murphy-Ullrich
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Adhesive Strength ◽  
Focal Adhesions ◽  
Lipid Product ◽  
Phosphoinositide 3 Kinase ◽  
Triton X ◽  
Adhesion Plaque ◽  
Adhesion Complex

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of α-actinin and vinculin from the focal adhesion complex to the Triton X-100–soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P3 binds to α-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of α-actinin with the integrin β subunit, and that PtdIns (3,4,5)-P3 could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P3, has important implications for the regulation of cellular adhesive strength and motility.

scholarly journals Physical confinement signals regulate the organization of stem cells in three dimensions

2016 ◽  
Vol 13 (123) ◽  
pp. 20160613 ◽  
Author(s):  
Sebastian V. Hadjiantoniou ◽  
David Sean ◽  
Maxime Ignacio ◽  
Michel Godin ◽  
Gary W. Slater ◽  
...  
Keyword(s):  
Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Interactions ◽  
Three Dimensions ◽  
Cell Substrate ◽  
Cell Cell

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro , mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells.

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