F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation.
Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca(2+) signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca(2+) signaling.
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Alexander
A
Babich
Shuixing
S
Li
Roddy S
RS
O'Connor
Michael C
MC
Milone
Bruce D
BD
Freedman
Janis K
JK
Burkhardt
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism*; D058105:Polymerization; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics / Q000378:metabolism
D000199:Actins; D011948:Receptors, Antigen, T-Cell; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D051966:Phospholipase C gamma; D024507:Nonmuscle Myosin Type IIA