Functional phosphatome requirement for protein homeostasis, networked mitochondria, and sarcomere structure in C. elegans muscle

Abstract

BackgroundSkeletal muscle is central to locomotion and metabolic homeostasis. The laboratory worm Caenorhabditis elegans has been developed into a genomic model for assessing the genes and signals that regulate muscle development and protein degradation. Past work has identified a receptor tyrosine kinase signalling network that combinatorially controls autophagy, nerve signal to muscle to oppose proteasome‐based degradation, and extracellular matrix‐based signals that control calpain and caspase activation. The last two discoveries were enabled by following up results from a functional genomic screen of known regulators of muscle. Recently, a screen of the kinome requirement for muscle homeostasis identified roughly 40% of kinases as required for C. elegans muscle health; 80 have identified human orthologues and 53 are known to be expressed in skeletal muscle. To complement this kinome screen, here, we screen most of the phosphatases in C. elegans . MethodsRNA interference was used to knockdown phosphatase‐encoding genes. Knockdown was first conducted during development with positive results also knocked down only in fully developed adult muscle. Protein homeostasis, mitochondrial structure, and sarcomere structure were assessed using transgenic reporter proteins. Genes identified as being required to prevent protein degradation were also knocked down in conditions that blocked proteasome or autophagic degradation. Genes identified as being required to prevent autophagic degradation were also assessed for autophagic vesicle accumulation using another transgenic reporter. Lastly, bioinformatics were used to look for overlap between kinases and phosphatases required for muscle homeostasis, and the prediction that one phosphatase was required to prevent mitogen‐activated protein kinase activation was assessed by western blot.ResultsA little over half of all phosphatases are each required to prevent abnormal development or maintenance of muscle. Eighty‐six of these phosphatases have known human orthologues, 57 of which are known to be expressed in human skeletal muscle. Of the phosphatases required to prevent abnormal muscle protein degradation, roughly half are required to prevent increased autophagy.ConclusionsA significant portion of both the kinome and phosphatome are required for establishing and maintaining C. elegans muscle health. Autophagy appears to be the most commonly triggered form of protein degradation in response to disruption of phosphorylation‐based signalling. The results from these screens provide measurable phenotypes for analysing the combined contribution of kinases and phosphatases in a multi‐cellular organism and suggest new potential regulators of human skeletal muscle for further analysis.

Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

“…PP2A phosphatase localizes to centrosomes and connects to SPD-5 indirectly through the adapter proteins RSA-1 and RSA-2 ( Schlaitz et al, 2007 ). Depletion of the catalytic subunit LET-92 causes pleiotropic effects such as reduced microtubule stability, mitotic spindle collapse, and increased autophagy, which could indirectly affect PCM disassembly ( Lehmann et al, 2017 ; Schlaitz et al, 2007 ). PP2A phosphatases function as holoenzymes comprising an invariant catalytic and structural subunit coupled to variable regulatory subunits that determine substrate specificity ( Janssens and Goris, 2001 ).…”

Greater loss of mitochondrial function with ageing is associated with earlier onset of sarcopenia in C. elegans

“…Recently large scale studies using RNAi have been conducted to investigate how muscle health is maintained in C. elegans [16–19]. These studies have examined the effect of knocking down more than 850 genes (approximately 4% of the C. elegans genome) on sub-cellular muscle architecture.…”