Nonmuscle Myosin II Isoforms Coassemble in Living Cells

Wednesday, September 24, 2014 — Poster Session IV
10:00 a.m. –12:00 p.m.	FAES Academic Center	NHLBI	CELLBIO-2

* FARE Award Winner

Authors

• J. R. Beach
• L. Shao
• K. Remmert
• D. Li
• E Betzig
• J. A. Hammer 3rd

Abstract

Nonmuscle myosin II (NMII) powers myriad developmental and cellular processes. To function, NMII monomers assemble into bipolar filaments that produce contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NMII (NMIIA, IIB and IIC), each supporting unique, as well as redundant, cellular functions. It remains unclear if NMII isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or filaments consisting entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged NMIIA, NMIIB and NMIIC, isoform-specific immunostaining of endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual NM II bipolar filaments inside cells. We show that NMII isoforms form heterotypic filaments in various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NMIIA and NMIIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. This differential distribution is less pronounced in freshly-spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NMII isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NMII isoforms.

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