SH3KBP1 scaffolds endoplasmic reticulum and controls skeletal myofibers architecture and integrity

Abstract

22 23 Myonuclei are actively positioned throughout muscular development. Guiraud, Christin, Couturier et al show that 24 SH3KBP1 scaffolds the ER through Calnexin interaction and controls myonuclei motion during early steps of 25 muscle fibers formation. Besides SH3KBP1 participates in cell fusion and T-tubules formation/maintenance in 26 mature skeletal muscle fibers and contributes to slow-down CNM-like phenotypes. Abstract 29 30 The building block of skeletal muscle is the multinucleated muscle fiber, formed by the fusion of hundreds of 31 mononucleated precursor cells, myoblasts. In the normal course of muscle fiber development or regeneration, 32 42 and maintenance of Sarcoplasmic Reticulum (SR) and Transverse-tubules (T-tubules). We also demonstrate that 43 in muscle fibers, GTPase dynamin-2 (DNM2) binds to SH3 domains of SH3KBP1. Interestingly, we observed that 44 Sh3kbp1 mRNA is up regulated in a mouse model harboring the most frequent mutation for Autosomal Dominant 45 CentroNuclear Myopathy (AD-CNM): Dnm2 +/R465W . SH3KBP1 thus appears as a compensation mechanism in this 46 CNM model since its depletion contributes to an increase of CNM-like phenotypes (reduction of muscle fibers 47 Cross-section Areas (CSA) and increase in slow fibers content). 48 Altogether our results identify SH3KBP1 as a new regulator of myonuclear domains establishment in the early 49 phase of muscle fibers formation through ER scaffolding and later in myofibers integrity through T-tubules 50 scaffolding/maintenance. 51 52 65 2012) and molecular motors, including dynein and kinesins (Gache et al, 2017). This dynamic of myonuclei 66 contributes to precocious alignment of myonuclei in immature fibers. Although comprehension of mechanisms 67 involved in myonuclear positioning during myofibers formation has recently progressed, how mispositioning of 68 myonuclei affects muscle function still remains an open question. 69 Abnormal myonuclei internalization in muscle fibers that are not directly linked to excessive regenerative 70 process is the hallmark of a group of humans myopathies called Centronuclear Myopathies (CNMs)(Romero & 71 605 606 Supplementary video 1 & 2: Timelapse experiments of primary myoblasts co-transfected with Lamin-607 Chromobody ® -RFP plasmids and with either Scramble-siRNA (Supplementary video-1) or Sh3kbp1-siRNA 608 (Supplementary video-2) and induce to form myotubes during 5 days after starting differentiation process. 609 Primary myotubes were recorded every 20 minutes for a period of time of 16 hours. 610 611 Materials and methods 612 17 613 Cell culture 614Primary myoblasts were collected from wild type C57BL6 mice as described before (Falcone et al, 2014; Pimentel et 615 al, 2017). Briefly, Hindlimb muscles from 6 days pups were extracted and digested with collagenase (Sigma, C9263-616 1G) and dispase (Roche, 04942078001). After a pre-plating step to discard contaminant cells such as fibroblasts, 617 myoblasts were cultured on matrigel coated-dish (Corning, 356231) and induced to differentiate in myot...