TY - JOUR
T1 - Small, Dynamic Clusters of Tir-Intimin Seed Actin Polymerization
AU - Oh, Dongmyung
AU - Liu, Xuyao
AU - Sheetz, Michael P.
AU - Kenney, Linda J.
N1 - Funding Information:
D.O. and X.L. contributed equally to this work. The authors would like to express their gratitude to Zhongwen Chen (Fudan University, China) and Mrinal Shah for their invaluable contributions to the SLB and EPEC pedestals, respectively. The authors also thank Prof. Gad Frankel (Imperial College London, UK) for generously providing EPEC mutant strains. Thanks also go to the MBI core facilities particularly the Protein purification core and the Science Communication Core, for providing purified proteins and illustrations. This work was supported by the Mechanobiology Institute at the National University of Singapore and the University of Texas Medical Branch, Galveston, Texas.
Publisher Copyright:
© 2023 Wiley-VCH GmbH.
PY - 2023
Y1 - 2023
N2 - The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.
AB - The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.
KW - enteropathogenic Escherichia coli
KW - in vivo single-molecule kinetics
KW - phase separation
KW - single-particle tracking photoactivated localization microscopy
KW - Tir clustering
UR - http://www.scopus.com/inward/record.url?scp=85169161338&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85169161338&partnerID=8YFLogxK
U2 - 10.1002/smll.202302580
DO - 10.1002/smll.202302580
M3 - Article
C2 - 37649226
AN - SCOPUS:85169161338
SN - 1613-6810
JO - Small
JF - Small
ER -