Abstract
Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation–contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM.
Highlights
Introduction of centronuclear myopathy (CNM) mutations intoBIN1 and dynamin 2Entry clones of mutant BIN1 in Src homology 3 (SH3) domain (BIN1Δ434–454, BIN1Δ435–454 and BIN1Δ436–454) were prepared by B-P recombination reaction of PCR products amplified from pEGFPmAmph2 using corresponding primers (Table S1) into pDONR201
Our results suggest that aberrant regulation of membrane remodeling by BIN1-dynamin 2 complex is tightly linked to the pathogenesis of CNM
The protein encoded by an autosomal recessive CNM mutant of BIN1 harboring a nonsense mutations in the SH3 domain, p.K575* (p.K436* in isoform 8), had previously been shown to have reduced binding affinity with dynamin 2 [3]. To examine if this alteration in the interaction between BIN1 and dynamin 2 is responsible for CNM pathogenesis, we examined tubule-like structures (TLS) arising from the overexpression of CNM mutant BIN1 in SH3 domain (Fig. 3A)
Summary
Received for publication, July 11, 2020, and in revised form, November 10, 2020 Published, Papers in Press, November 13, 2020, https://doi.org/10.1074/jbc.RA120.015184 Kenshiro Fujise1, Mariko Okubo2,3 , Tadashi Abe1, Hiroshi Yamada1, Ichizo Nishino2 , Satoru Noguchi2, Kohji Takei1,*, and Tetsuya Takeda1,* From the 1Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan; 2National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan; and 3Department of Pediatrics, The University of Tokyo, Tokyo, Japan
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