Biology subjects

Benian, G. M.

Publications and source records attributed to Benian, G. M..

8 recordsLinked to original sources

bioRxiv2025

The Lipid-binding PX Domain of RRC-1 (ARHGAP32/33) is Required for Optimal Assembly and Function of Integrin Adhesion Complexes at the Muscle Cell Boundary in C. elegans

Integrin adhesion complexes (IACs) are a network of many proteins that serve as anchors of the cell to the extracellular matrix (ECM). In muscle, IACs located at costameres, also serve to transmit the force of muscle contraction to the outside of the cell. We have reported that IACs, which are found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the RacGEF PIX-1 for their proper assembly or maintenance. We have reported that a RacGAP for the PIX pathway is RRC-1, is in a complex with PIX-1, and that RRC-1 is required for assembly or maintenance of IACs at MCBs. Our previous studies suggested that RRC-1 might be associated with the muscle cell membrane, and here we present evidence that this occurs via its PX domain, a domain that is known to bind to membrane phosphoinositides (PIPs). We predict the existence of a PX domain based on bioinformatic analysis and AlphaFold3, which includes conserved residues characteristic of most PX domains and a PIP binding site. This region of RRC-1 binds to phosphoinositides in vitro. Analysis of a nematode strain that has an in-frame deletion of the PX domain, indicates that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffold protein GIT-1. Lastly, we show that the overexpression of the full length RRC-1, but not RRC-1 with an in-frame deletion of its PX domain, results in reduced accumulation of IAC components and reduced whole animal movement. Our study highlights the importance of RRC-1s lipid interactions at the cell membrane for proper assembly and function of IACs in C. elegans muscle. Article SummaryIntegrin adhesion complexes (IACs) facilitate the transmission of force of muscle contraction to the outside of the cell. IACs, found at the bases of dense bodies and M-lines, and at muscle cell boundaries (MCB) in C. elegans muscle, require the PIX-1 (a RacGEF) signaling pathway for their assembly or maintenance. A RacGAP for the PIX pathway is RRC-1. Here, we show that RRC-1 has a PX domain that binds to membrane phosphoinositides. We also show that normal localization of RRC-1 to the MCB requires both its PX domain and the PIX scaffolding protein GIT-1.

cell biology↗

bioRxiv2025

IPMK-1 (inositol phosphate multikinase) is required for optimal integrin adhesion complex assembly in C. elegans muscle

Integrin adhesion complexes (IACs) are a network of many proteins including the transmembrane protein integrin that anchor cells to the extracellular matrix (ECM). In C. elegans muscle IACs exist at the bases of dense bodies and M-lines, and at the boundaries of adjacent muscle cells (MCBs). We have reported that the proper assembly of IACs requires the Rac GEF PIX-1 and members of the PIX-1 pathway. Here we report that further investigation into the original Million Mutation Project strain that led us to uncover the role of PIX-1 in muscle, revealed a genetic enhancer of the PIX-1 phenotype. Genetic mapping shows that the enhancer is a recessive mutation in a single gene, ipmk-1. ipmk-1, encodes inositol phosphate multikinase and converts PIP2 to PIP3, IP3 to IP4, and IP4 to IP5. Loss of function of pix-1 results in reduced accumulation of IAC proteins at the MCB, and ipmk-1; pix-1 double mutants additionally display a large gap between adjacent muscle cells. ipmk-1 similarly enhances other members of the PIX pathway including PAK-1, and the RacGAP RRC-1. Lack of ipmk-1 itself results in abnormal clumping of IAC components at the MCB, decreased whole animal locomotion, and mild sarcomere disorganization. Analysis of ipmk-1; ipp-5 double mutants suggest that the MCB defect of ipmk-1 is due to decreased PIP3 in the cell membrane rather than the decreased IP3 and Ca2+ signaling. Overall, these studies provide in vivo evidence, specifically in muscle, that the level of PIP3 and PIP2 influence the proper assembly of IACs. Article SummaryC. elegans muscle have integrin adhesion complexes (IACs) at dense bodies, M-lines, and boundaries between adjacent muscle cells (MCBs). The Benian lab reported that assembly of these IACs requires the PIX-1 pathway. Here they report ipmk-1 is a genetic enhancer of the PIX pathway. ipmk-1encodes inositol phosphate multikinase converting PIP2 to PIP3. pix-1 shows reduced accumulation of IAC components; ipmk-1; pix-1 double mutants additionally have large gaps between muscle cells. ipmk-1 mutants display abnormal clumping of IAC components. This is the first report of the importance of these lipids in assembly of IACs in vivo, and in particular, in muscle.

cell biology↗

bioRxiv2024

Age-dependent Changes in a Chaperone Complex in the Mouse Heart

The exquisitely organized sarcomere, the unit of contraction of striated muscle, is a stable structure with slow turnover of its components. The myosin chaperone UNC-45 and its binding partners, Hsp90 and Hsp70, are required for the initial folding of the myosin head domain and the assembly of myosin into thick filaments. There is increasing evidence that the UNC-45 system has an important role during aging to preserve sarcomere organization. Its decline may be a key factor in sarcopenia. Unlike skeletal muscle, the UNC-45 system in cardiac muscle in aging heart has not been examined extensively. Here we show that Unc45b and Hsp70 are localized to sarcomeric Z-discs in the mouse heart. We further show that during aging, there is a decline in the levels of myosin heavy chain, Unc-45b and Hsp70, but not Hsp90. While the decrease in Unc45b appears to be at the mRNA level, the decrease in the levels of myosin and Hsp70 were not at the mRNA but at the protein level. We have reported that in skeletal muscle, there is a decline in both Unc45b and Hsp90, and here we show that there is no such decline of Hsp70 in skeletal muscle. Hsp70 levels also did not decline with age in the brain or the liver. This heart-specific decrease of Hsp70 through its function as an Unc45b/Hsp70 complex might account for the age-dependent worsening of cardiomyopathies, and through Hsp70s multiple Unc-45b-independent functions, affect the folding and assembly of many other proteins in the aging heart.

cell biology↗

bioRxiv2023

FARL-11 (STRIP1/2) is Required for Sarcomere and Sarcoplasmic Reticulum Organization in C. elegans

Protein phosphatase 2A (PP2A) functions in a variety of cellular contexts. PP2A can assemble into four different complexes based on the inclusion of different regulatory or targeting subunits. The B regulatory subunit "striatin" forms the STRIPAK complex consisting of striatin, a catalytic subunit (PP2AC), striatin interacting protein 1 (STRIP1), and MOB family member 4 (MOB4). In yeast and C. elegans, STRIP1 is required for formation of the endoplasmic reticulum (ER). Since the sarcoplasmic reticulum (SR) is the highly organized muscle-specific version of ER, we sought to determine the function of the STRIPAK complex in muscle using C. elegans. CASH-1 (striatin) and FARL-11 (STRIP1/2) form a complex in vivo, and each protein is localized to SR. Missense mutations and single amino acid losses in farl-11 and cash-1 each result in similar sarcomere disorganization. A missense mutation in farl-11 shows no detectable FARL-11 protein by immunoblot, disruption of SR organization around M-lines, and altered levels of the SR Ca+2 release channel UNC-68. SummaryProtein phosphatase 2A forms a STRIPAK complex when it includes the targeting B subunit "striatin" and STRIP1. STRIP1 is required for formation of ER. We show that in muscle STRIP1 is required for organization of SR and sarcomeres.

cell biology↗

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↗

bioRxiv2021

Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.

molecular biology↗

bioRxiv2021

Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic C. elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signalling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g zebrafish).

biophysics↗

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↗