Moody, J. C. | Search bioRxiv

bioRxiv2022

The Rho GAP RRC-1 is a member of the PIX pathway and controls assembly or stability of integrin adhesion complexes in muscle

GTPases cycle between active GTP bound and inactive GDP bound forms. Exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). GTPase activating proteins (GAPs) accelerate GTP hydrolysis, to promote the GDP bound form. Recently, we reported that the GEF called PIX is required for assembly or stability of integrin adhesion complexes (IAC) in striated muscle. A GAP for the PIX pathway had not been identified in any cell type or organism. A screen in C. elegans of mutants in 18 proteins containing Rho GAP domains and expressed in muscle revealed that loss of function of rrc-1 results in loss of IAC components at the muscle cell boundary (MCB). RRC-1 contains an SH3 domain and a Rho GAP domain, and is localized to the IACs of MCBs, like PIX-1. rrc-1 mutants show reduced accumulation of IAC components at the MCB, sarcomere disorganization, and reduced whole animal locomotion. Knockdown of git-1, which encodes a PIX scaffold protein, reduces the level of RRC-1. Localization at MCBs of RRC-1 depends on pix-1, and the localization of PIX-1 depends on rrc-1. These results suggest that RRC-1 is a RhoGAP for the PIX pathway in muscle.

cell biology↗

bioRxiv2020

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

In C. elegans, unc-89 encodes a set of giant multi-domain proteins (up 8,081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647-742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain "interkinase region" has low sequence complexity, high proline content and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals express normal levels of giant internally deleted UNC-89 proteins, and yet show severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generated in-frame deletion to contain an HA tag at the N-terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake. HighlightsO_LIThe giant muscle proteins UNC-89 and its mammalian homologs have an [~]700 aa non-domain region lying between two protein kinase domains C_LIO_LIBy single molecule force spectroscopy UNC-89 non-domain region is an elastic random coil C_LIO_LINematodes lacking this non-domain region have disorganized sarcomeres and reduced whole animal locomotion C_LIO_LIUNC-89 non-domain region is required for proper assembly of A-bands from thick filaments C_LI

cell biology↗