Cell adaptive response to extracellular matrix density is controlled by ICAP-1–dependent β1-integrin affinity

Cell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially understood. We provide evidence for a novel mechanistic insight into focal adhesion (FA) dynamics by demonstrating that integrin cytoplasmic domain–associated protein 1 (ICAP-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1–deficient mouse embryonic fibroblasts and cells expressing active β1 integrin, shows that the integrin high affinity state favored by talin is antagonistically controlled by ICAP-1. This affinity switch results in modulation in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the ICAP-1–dependent decrease in integrin affinity allows cell sensing of matrix surface density, suggesting that integrin conformational changes are important in mechanotransduction. Our results clarify the function of ICAP-1 in cell adhesion and highlight the central role it plays in the cell's integrated response to the extracellular microenvironment.

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Traffic watch: Live-cell imaging

The Biochemist ◽

10.1042/bio02503015 ◽

2003 ◽

Vol 25 (3) ◽

pp. 15-17

Author(s):

David J. Stephens

Keyword(s):

Electron Microscopy ◽

Cell Biology ◽

Live Cell Imaging ◽

Cell Imaging ◽

Living Cells ◽

Robert Hooke ◽

The Core ◽

Biology Research ◽

Insight Into ◽

Dynamics Process

Microscopy has been at the core of cell biology research ever since the coining of the term ‘cell’ by Robert Hooke in the 17th Century1. For many years, it has been possible to gain insight into ‘steady-state’ cellular function from the analysis of fixed samples, but it is only relatively recently that imaging of living cells has become a widely used tool to support biochemical and electron microscopy studies. Membrane traffic research, which by its very nature is a highly dynamics process, has benefited hugely from the ability to image specific processes in living cells and tissues.

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Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

10.1101/2022.01.05.475028 ◽

2022 ◽

Author(s):

Yohei Kono ◽

Stephen A. Adam ◽

Karen Reddy ◽

Yixian Zheng ◽

Ohad Medalia ◽

...

Keyword(s):

Nuclear Envelope ◽

Live Cell Imaging ◽

Cell Imaging ◽

Nuclear Lamina ◽

Structural Components ◽

Cell Nuclei ◽

Embryonic Fibroblasts ◽

Nuclear Lamins ◽

Laser Microirradiation

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

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Brain and Breast Cancer Cells with PTEN Loss of Function Demonstrate Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Using 3D Printed Scaffolds and Aligned Microfiber Tracts

10.21203/rs.3.rs-826338/v1 ◽

2021 ◽

Author(s):

Annalena Wieland ◽

Pamela L. Strissel ◽

Hannah Schorle ◽

Ezgi Bakirci ◽

Dieter Janzen ◽

...

Keyword(s):

Breast Cancer ◽

Cell Morphology ◽

Live Cell Imaging ◽

Essential Role ◽

Cell Imaging ◽

Loss Of Function ◽

Pten Loss ◽

3D Printed ◽

And Migration

Abstract Background: Glioblastoma multiforme (GBM) and triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with very poor patient outcomes. GBM uses axons and vessels as migratory cues to disseminate, however it is not known, if TNBC shares the same behavior. There is a need to understand brain tumor cell spreading and if GBM and TNBC have similar migration properties involving the signaling pathway RHOB-ROCK-PTEN. We tested for durotaxis, adherence and migration of GBM and TNBC using live-cell imaging and performed molecular analyses on three-dimensional (3D) structures.Methods: Aligned 3D printed scaffolds and microfibers were designed to mimic brain axon tracts and vessels for migration. GBM and TNBC cell lines, each with opposing PTEN genotypes, were analysed with RHO, ROCK and PTEN inhibitors and rescuing PTEN function using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while SEM, confocal microscopy and cell tracking addressed cell morphology. Results: GBM and TNBC with homozygote PTEN loss of function and RHOB high expression were amoeboid shaped and demonstrated enhanced durotaxis, adhesion and migration on 3D microfibers, in contrast to PTEN wildtype GBM and TNBC showing elongated cells and low RHOB. RNA-sequencing exhibited that RHOB was significantly the highest expressed gene in GBM PTEN loss of function cells. Pathway inhibitors and PTEN rescue of function verified an essential role of RHOB-ROCK-PTEN signaling for durotaxis, adhesion, migration, cell morphology and plasticity using 3D printed microfibers. Conclusions: This study validates a significant role of a PTEN genotype for cellular properties including durotaxis, adhesion and migration. GBM and TNBC cells with PTEN loss of function have a greater affinity for stiffer brain structures promoting metastasis. We propose the significance of PTEN and RHOB in cellular oncology not only for primary tumors, but also for metastasizing tumors, where RHOB inhibitors could play an essential role for improved therapy.

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GMFβ controls branched actin content and lamellipodial retraction in fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.201501094 ◽

2015 ◽

Vol 209 (6) ◽

pp. 803-812 ◽

Cited By ~ 21

Author(s):

Elizabeth M. Haynes ◽

Sreeja B. Asokan ◽

Samantha J. King ◽

Heath E. Johnson ◽

Jason M. Haugh ◽

...

Keyword(s):

Cell Migration ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Directional Migration ◽

Pharmacological Inhibition ◽

Primary Activity ◽

And Migration ◽

Important Structure

The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFβ, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFβ decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.

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Protein 4.1G Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryonic Fibroblasts through the β1 Integrin Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m115.658591 ◽

2015 ◽

Vol 291 (5) ◽

pp. 2170-2180 ◽

Cited By ~ 2

Author(s):

Lixiang Chen ◽

Ting Wang ◽

Yaomei Wang ◽

Jingxin Zhang ◽

Yuanming Qi ◽

...

Keyword(s):

Cell Adhesion ◽

Β1 Integrin ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

And Migration

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Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

The Journal of Cell Biology ◽

10.1083/jcb.200705146 ◽

2007 ◽

Vol 179 (4) ◽

pp. 635-641 ◽

Cited By ~ 22

Author(s):

Boris M. Slepchenko ◽

Irina Semenova ◽

Ilya Zaliapin ◽

Vladimir Rodionov

Keyword(s):

Actin Filaments ◽

Live Cell Imaging ◽

Intracellular Transport ◽

Cell Imaging ◽

Major Change ◽

Living Cells ◽

Transport Systems ◽

Switching Rate ◽

Novel Approach ◽

Insight Into

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.

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Assembly of multiprotein complexes that control genome function

The Journal of Cell Biology ◽

10.1083/jcb.200811080 ◽

2009 ◽

Vol 185 (1) ◽

pp. 21-26 ◽

Cited By ~ 36

Author(s):

Christoffel Dinant ◽

Martijn S. Luijsterburg ◽

Thomas Höfer ◽

Gesa von Bornstaedt ◽

Wim Vermeulen ◽

...

Keyword(s):

Mathematical Modeling ◽

Dna Repair ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Imaging Studies ◽

Multiprotein Complexes ◽

Insight Into

Live-cell imaging studies aided by mathematical modeling have provided unprecedented insight into assembly mechanisms of multiprotein complexes that control genome function. Such studies have unveiled emerging properties of chromatin-associated systems involved in DNA repair and transcription.

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Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

Journal of Virology ◽

10.1128/jvi.01327-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 4

Author(s):

Eric N. Salgado ◽

Brian Garcia Rodriguez ◽

Nagarjun Narayanaswamy ◽

Yamuna Krishnan ◽

Stephen C. Harrison

Keyword(s):

Conformational Changes ◽

Outer Layer ◽

Live Cell Imaging ◽

Molecular Mechanisms ◽

Cell Imaging ◽

Tight Binding ◽

Live Cell ◽

Membrane Disruption ◽

Molecular Events

ABSTRACTBound calcium ions stabilize many nonenveloped virions. Loss of Ca2+from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca2+in vitrostrips away both outer layer proteins; we and others have postulated that the loss of Ca2+triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+sensor, the timing of Ca2+loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca2+sensor, placed on VP5*, monitors the Ca2+concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca2+precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCENonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca2+sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+binding stabilizes many other viruses of eukaryotes, and Ca2+loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.

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