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1-13 of 13
Martin J. Humphries
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Journal Articles
Megan R. Chastney, Craig Lawless, Jonathan D. Humphries, Stacey Warwood, Matthew C. Jones, David Knight, Claus Jorgensen, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2020) 219 (8): e202003038.
Published: 25 June 2020
Abstract
Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.
Includes: Supplementary data
Journal Articles
John G. Lock, Francesco Baschieri, Matthew C. Jones, Jonathan D. Humphries, Guillaume Montagnac, Staffan Strömblad, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2019) 218 (7): 2086–2095.
Published: 17 June 2019
Abstract
An understanding of the mechanisms whereby cell adhesion complexes (ACs) relay signals bidirectionally across the plasma membrane is necessary to interpret the role of adhesion in regulating migration, differentiation, and growth. A range of AC types has been defined, but to date all have similar compositions and are dependent on a connection to the actin cytoskeleton. Recently, a new class of AC has been reported that normally lacks association with both the cytoskeleton and integrin-associated adhesome components, but is rich in components of the clathrin-mediated endocytosis machinery. The characterization of this new type of adhesion structure, which is emphasized by mitotic cells and cells in long-term culture, identifies a hitherto underappreciated link between the adhesion machinery and clathrin structures at the plasma membrane. While this discovery has implications for how ACs are assembled and disassembled, it raises many other issues. Consequently, to increase awareness within the field, and stimulate research, we explore a number of the most significant questions below.
Journal Articles
Journal:
Journal of Cell Biology
Journal of Cell Biology (2018) 217 (9): 3203–3218.
Published: 21 June 2018
Abstract
In most tissues, anchorage-dependent growth and cell cycle progression are dependent on cells engaging extracellular matrices (ECMs) via integrin–receptor adhesion complexes. In a highly conserved manner, cells disassemble adhesion complexes, round up, and retract from their surroundings before division, suggestive of a primordial link between the cell cycle machinery and the regulation of cell adhesion to the ECM. In this study, we demonstrate that cyclin-dependent kinase 1 (CDK1) mediates this link. CDK1, in complex with cyclin A2, promotes adhesion complex and actin cytoskeleton organization during interphase and mediates a large increase in adhesion complex area as cells transition from G1 into S. Adhesion complex area decreases in G2, and disassembly occurs several hours before mitosis. This loss requires elevated cyclin B1 levels and is caused by inhibitory phosphorylation of CDK1–cyclin complexes. The inactivation of CDK1 is therefore the trigger that initiates remodeling of adhesion complexes and the actin cytoskeleton in preparation for rapid entry into mitosis.
Includes: Supplementary data
Journal Articles
Edward R. Horton, Jonathan D. Humphries, Ben Stutchbury, Guillaume Jacquemet, Christoph Ballestrem, Simon T. Barry, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2016) 212 (3): 349–364.
Published: 01 February 2016
Abstract
Integrin adhesion complexes (IACs) form mechanochemical connections between the extracellular matrix and actin cytoskeleton and mediate phenotypic responses via posttranslational modifications. Here, we investigate the modularity and robustness of the IAC network to pharmacological perturbation of the key IAC signaling components focal adhesion kinase (FAK) and Src. FAK inhibition using AZ13256675 blocked FAK Y397 phosphorylation but did not alter IAC composition, as reported by mass spectrometry. IAC composition was also insensitive to Src inhibition using AZD0530 alone or in combination with FAK inhibition. In contrast, kinase inhibition substantially reduced phosphorylation within IACs, cell migration and proliferation. Furthermore using fluorescence recovery after photobleaching, we found that FAK inhibition increased the exchange rate of a phosphotyrosine (pY) reporter (dSH2) at IACs. These data demonstrate that kinase-dependent signal propagation through IACs is independent of gross changes in IAC composition. Together, these findings demonstrate a general separation between the composition of IACs and their ability to relay pY-dependent signals.
Includes: Multimedia, Supplementary data
Journal Articles
Guillaume Jacquemet, David M. Green, Rebecca E. Bridgewater, Alexander von Kriegsheim, Martin J. Humphries, Jim C. Norman, Patrick T. Caswell
Journal:
Journal of Cell Biology
Journal of Cell Biology (2013) 202 (6): 917–935.
Published: 09 September 2013
Abstract
Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.
Includes: Supplementary data
Journal Articles
Daniel C. Worth, Kairbaan Hodivala-Dilke, Stephen D. Robinson, Samantha J. King, Penny E. Morton, Frank B. Gertler, Martin J. Humphries, Maddy Parsons
Journal:
Journal of Cell Biology
Journal of Cell Biology (2010) 189 (2): 369–383.
Published: 19 April 2010
Abstract
Integrins are fundamental to the control of protrusion and motility in adherent cells. However, the mechanisms by which specific members of this receptor family cooperate in signaling to cytoskeletal and adhesion dynamics are poorly understood. Here, we show that the loss of β3 integrin in fibroblasts results in enhanced focal adhesion turnover and migration speed but impaired directional motility on both 2D and 3D matrices. These motility defects are coupled with an increased rate of actin-based protrusion. Analysis of downstream signaling events reveals that loss of β3 integrin results in a loss of protein kinase A–dependent phosphorylation of the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP). Dephosphorylated VASP in β3-null cells is preferentially associated with Rap1-GTP–interacting adaptor molecule (RIAM) both in vitro and in vivo, which leads to enhanced formation of a VASP–RIAM complex at focal adhesions and subsequent increased binding of talin to β1 integrin. These data demonstrate a novel mechanism by which αvβ3 integrin acts to locally suppress β1 integrin activation and regulate protrusion, adhesion dynamics, and persistent migration.
Includes: Supplementary data
Journal Articles
Janet A. Askari, Christopher J. Tynan, Stephen E.D. Webb, Marisa L. Martin-Fernandez, Christoph Ballestrem, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2010) 188 (6): 891–903.
Published: 15 March 2010
Abstract
Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.
Includes: Supplementary data
Journal Articles
Journal:
Journal of Cell Biology
Journal of Cell Biology (2009) 185 (7): 1275–1284.
Published: 29 June 2009
Abstract
Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin–ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin α 5 β 1 -Fc fusion protein or membrane α 5 β 1 . Force prolonged bond lifetimes in the 10–30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca 2+ /Mg 2+ to Mg 2+ /EGTA and to Mn 2+ caused longer lifetime in the same 10–30-pN catch bond region. A truncated α 5 β 1 construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.
Includes: Supplementary data
Journal Articles
Mark D. Bass, Mark R. Morgan, Kirsty A. Roach, Jeffrey Settleman, Andrew B. Goryachev, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2008) 181 (6): 1013–1026.
Published: 09 June 2008
Abstract
The fibronectin receptors α 5 β 1 integrin and syndecan-4 cocluster in focal adhesions and coordinate cell migration by making individual contributions to the suppression of RhoA activity during matrix engagement. p190Rho–guanosine triphosphatase–activating protein (GAP) is known to inhibit RhoA during the early stages of cell spreading in an Src-dependent manner. This paper dissects the mechanisms of p190RhoGAP regulation and distinguishes the contributions of α 5 β 1 integrin and syndecan-4. Matrix-induced tyrosine phosphorylation of p190RhoGAP is stimulated solely by engagement of α 5 β 1 integrin and is independent of syndecan-4. Parallel engagement of syndecan-4 causes redistribution of the tyrosine-phosphorylated pool of p190RhoGAP between membrane and cytosolic fractions by a mechanism that requires direct activation of protein kinase C α by syndecan-4. Activation of both pathways is necessary for the efficient regulation of RhoA and, as a consequence, focal adhesion formation. Accordingly, we identify p190RhoGAP as the convergence point for adhesive signals mediated by α 5 β 1 integrin and syndecan-4. This molecular mechanism explains the cooperation between extracellular matrix receptors during cell adhesion.
Includes: Supplementary data
Journal Articles
In Special Collection:
JCB65: Cell Adhesion and Migration
Jonathan D. Humphries, Pengbo Wang, Charles Streuli, Benny Geiger, Martin J. Humphries, Christoph Ballestrem
Journal:
Journal of Cell Biology
Journal of Cell Biology (2007) 179 (5): 1043–1057.
Published: 03 December 2007
Abstract
Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.
Includes: Supplementary data
Journal Articles
Mark D. Bass, Kirsty A. Roach, Mark R. Morgan, Zohreh Mostafavi-Pour, Tobias Schoen, Takashi Muramatsu, Ulrike Mayer, Christoph Ballestrem, Joachim P. Spatz, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2007) 177 (3): 527–538.
Published: 07 May 2007
Abstract
Cell migration in wound healing and disease is critically dependent on integration with the extracellular matrix, but the receptors that couple matrix topography to migratory behavior remain obscure. Using nano-engineered fibronectin surfaces and cell-derived matrices, we identify syndecan-4 as a key signaling receptor determining directional migration. In wild-type fibroblasts, syndecan-4 mediates the matrix-induced protein kinase Cα (PKCα)–dependent activation of Rac1 and localizes Rac1 activity and membrane protrusion to the leading edge of the cell, resulting in persistent migration. In contrast, syndecan-4–null fibroblasts migrate randomly as a result of high delocalized Rac1 activity, whereas cells expressing a syndecan-4 cytodomain mutant deficient in PKCα regulation fail to localize active Rac1 to points of matrix engagement and consequently fail to recognize and respond to topographical changes in the matrix.
Includes: Supplementary data
Journal Articles
Ying Wei, Ralf-Peter Czekay, Liliane Robillard, Matthias C. Kugler, Feng Zhang, Kevin K. Kim, Jian-ping Xiong, Martin J. Humphries, Harold A. Chapman
Journal:
Journal of Cell Biology
Journal of Cell Biology (2005) 168 (3): 501–511.
Published: 31 January 2005
Abstract
Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with β1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the β-propeller of α3β1 empowers vitronectin adhesion by this integrin. How uPAR modifies other β1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds α5β1 and rather than blocking, renders fibronectin (Fn) binding by α5β1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of α5β1–uPAR to Fn type III repeats 12–15 in addition to type III repeats 9–11 bound by α5β1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall α5β1 conformation. A β1 peptide (res 224NLDSPEGGF232) that models near the known α-chain uPAR-binding region, or a β1-chain Ser227Ala point mutation, abrogated effects of uPAR on α5β1. Direct binding and regulation of α5β1 by uPAR implies a modified “bent” integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.
Includes: Supplementary data
Journal Articles
Zohreh Mostafavi-Pour, Janet A. Askari, Scott J. Parkinson, Peter J. Parker, Tony T.C. Ng, Martin J. Humphries
Journal:
Journal of Cell Biology
Journal of Cell Biology (2003) 161 (1): 155–167.
Published: 14 April 2003
Abstract
The fibronectin (FN)-binding integrins α4β1 and α5β1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of α4 + /α5 + A375-SM melanoma cell adhesion to fragments of FN that interact selectively with α4β1 and α5β1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, α5β1 and α4β1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; α5β1 requires a proteoglycan coreceptor (syndecan-4), and α4β1 does not. Second, adhesion via α5β1 caused an eightfold increase in protein kinase Cα (PKCα) activation, but only basal PKCα activity was observed after adhesion via α4β1. Pharmacological inhibition of PKCα and transient expression of dominant-negative PKCα, but not dominant-negative PKCδ or PKCζ constructs, suppressed focal adhesion formation and cell migration mediated by α5β1, but had no effect on α4β1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCα signaling as central to the functional differences between α4β1 and α5β1.