Waterman Clare M.

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Waterman
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Clare M.
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  • Article
    Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation
    (Rockefeller University Press, 2010-03-22) Pasapera, Ana M. ; Schneider, Ian C. ; Rericha, Erin ; Schlaepfer, David D. ; Waterman, Clare M.
    Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.
  • Article
    NLRP3 inflammasome assembly in neutrophils is supported by PAD4 and promotes NETosis under sterile conditions
    (Frontiers Media, 2021-03-28) Münzer, Patrick ; Negro, Roberto ; Fukui, Shoichi ; di Meglio, Lucas ; Aymonnier, Karen ; Chu, Long ; Cherpokova, Deya ; Gutch, Sarah ; Sorvillo, Nicoletta ; Shi, Lai ; Magupalli, Venkat Giri ; Weber, Alexander N. R. ; Scharf, Rüdiger E. ; Waterman, Clare M. ; Wu, Hao ; Wagner, Denisa D.
    Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.
  • Article
    Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration
    (Nature Publishing Group, 2017-12-11) Nordenfelt, Pontus ; Moore, Travis I. ; Mehta, Shalin B. ; Kalappurakkal, Joseph Mathew ; Swaminathan, Vinay ; Koga, Nobuyasu ; Lambert, Talley J. ; Baker, David ; Waters, Jennifer C. ; Oldenbourg, Rudolf ; Tani, Tomomi ; Mayor, Satyajit ; Waterman, Clare M. ; Springer, Timothy
    Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.
  • Article
    NLRP3 is essential for neutrophil polarization and chemotaxis in response to leukotriene B4 gradient
    (National Academy of Sciences, 2023-08-21) Van Bruggen, Stijn ; Jarrot, Pierre-Andre ; Thomas, Eline ; Sheehy, Casey E. ; Silva, Camila M. S. ; Hsu, Alan Y. ; Cunin, Pierre ; Nigrovic, Peter A. ; Gomes, Edgar R. ; Luo, Hongbo R. ; Waterman, Clare M. ; Wagner, Denisa D.
    Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1β and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3−/−) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3−/− neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.