HUM-7, an unconventional myosin with a RhoGAP domain, is required for assembly of integrin adhesion complexes in muscle Restricted; Files Only
Derossi, Stefano (Spring 2024)
Abstract
In vertebrate striated muscle (skeletal and cardiac), much of the force of muscle contraction is transmitted to the outside of the cell via costameres, which are muscle-specific integrin adhesion complexes (IACs). Costameres attach myofibrils located at the perimeter of the muscle cell to the muscle cell membrane and overlying extracellular matrix, and occur at each sarcomeric Z-disk. In the striated muscle of C. elegans, IACs reside at 3 locations—the bases of the sarcomeric M-lines and dense bodies (Z-disks) and at the muscle cell boundaries (MCBs). Each IAC consists of the heterodimeric transmembrane protein integrin and many proteins associated with it both intra- and extracellularly. The MCBs contain only a subset of proteins found at dense bodies. In a screen for mutants with defects in the MCB, we identified the gene pix-1, which encodes a RacGEF (guanine nucleotide exchange factor) (Moody et al., 2020) and the gene rrc-1, which encodes a RacGAP (GTPase activating protein) (Moody et al., 2024). During RacGAP screening, we also found that hum-7 mutants have defective MCBs. The HUM-7 protein is predicted to contain an RA (Ras association) domain, a myosin head domain, 4 consecutive IQ domains, 2 C1 (phorbol ester/diacylglycerol binding) domains, and a RhoGAP domain. Based on the sequence of its myosin motor domain and the presence of the other domains, HUM-7 is a class IX unconventional myosin. Two deletion mutants of hum-7 show less accumulation of IAC components at the MCB, similar to pix-1 and rrc-1. Two missense mutations in myosin head region show MCB defects, but one missense mutation in the RhoGAP domain did not show the defect in MCB, providing preliminary evidence that the myosin motor may be the domain critical for HUM-7’s MCB function. PIX-1 is still localized to MCBs in several hum-7 mutants, suggesting the HUM-7 may not be a component of the PIX signaling pathway. We prepared an antibody against HUM-7, and localized HUM-7 in muscle cells. We found that anti-HUM-7 antibodies localize between dense bodies and at the MCB but flanking the location of IAC components in body wall muscle cells. Transgenic rescue experiments are underway to determine which domain of HUM-7 more definitively is critical for its muscle function. Since most unconventional myosins deliver cargo along actin tracks, we hypothesize that HUM-7 functions as a “truck” to deliver IAC components towards the MCB and between dense body regions. As a first step to test this hypothesis, we performed yeast 2 hybrid assays to determine if the C-terminal half of HUM-7 interacts with any of the known components of the MCB. However, the results were negative. We are currently testing the hypothesis that an intermediary protein might be involved in attaching the HUM-7 motor to possible IAC cargo.
Table of Contents
Introduction……….…..1
Results and Discussion…………….5
Materials & Methods……………..21
References………………..24
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File download under embargo until 24 May 2025 | 2024-04-14 21:13:21 -0400 | File download under embargo until 24 May 2025 |
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