Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy
Human Molecular Genetics
Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy
Laura L. Smith 0
Vandana A. Gupta 0
Alan H. Beggs 0
0 Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School , Boston, MA 02115 , USA
Autosomal recessive centronuclear myopathy (CNM2), caused by mutations in bridging integrator 1 (BIN1), is a mildly progressive neuromuscular disorder characterized by abnormally centralized myonuclei and muscle weakness. BIN1 is important for membrane sensing and remodeling in vitro in different cell types. However, to fully understand the biological roles of BIN1 in vivo and to answer critical questions concerning the muscle-specific function of BIN1 in vertebrates, robust small animal models are required. In this study, we create and characterize a novel zebrafish model of CNM2 using antisense morpholinos. Immunofluorescence and histopathological analyses of Bin1-deficient zebrafish skeletal muscle reveal structural defects commonly reported in human CNM2 biopsies. Live imaging of zebrafish embryos shows defective calcium release in bin1 morphants, linking the presence of abnormal triads to impairments in intracellular signaling. RNA-mediated rescue assays demonstrate that knockdown of zebrafish bin1 can reliably examine the pathogenicity of novel BIN1 mutations in vivo. Finally, our results strongly suggest that the phosphoinositide-binding domain of BIN1, present only in skeletal muscle isoforms, may be more critical for muscle maturation and maintenance than for early muscle development. Overall, our data support that BIN1 plays an important role in membrane tubulation and may promote skeletal muscle weakness in CNM2 by disrupting machinery necessary for excitation - contraction coupling in vertebrate organisms. The reproducible phenotype of Bin1-deficient zebrafish, together with the generalized advantages of the teleost system, makes this model readily adaptable to high-throughput screening strategies and may be used to identify therapies for CNM2 and related neuromuscular diseases.
INTRODUCTION
Centronuclear myopathies (CNMs) are a heterogeneous group of
congenital disorders characterized by muscle weakness and
abnormal nuclear centralization in myofibers. Several genetic
forms have now been described, and vary in terms of age of
onset, severity of clinical symptoms and mode of inheritance.
Pathogenic CNM mutations are most commonly reported in the
phosphoinositide (PI) phosphatase myotubularin (MTM1) and
the large GTPase dynamin 2 (DNM2) genes, resulting in neonatal
X-linked (CNMX; MIM #310400) and autosomal dominant
(CNM1; MIM #160150) forms of the disease, respectively
(
1,2
). Variants in other genes, including the ryanodine receptor
calcium release channel (RYR1), titin (TTN) and
myotubularinrelated protein 14 (MTMR14), have also been identified in rare
cases with centralized nuclei on muscle biopsy (
3 – 6
). Autosomal
recessive CNM (CNM2; MIM #255200) is most often
associated with homozygous partial loss-of-function mutations
in the bridging integrator 1 (BIN1) gene (
7
). Compared with
MTM1- and DNM2-related CNM, pathological mechanisms
underlying BIN1-related CNM remain particularly unclear,
owing to fewer available human biopsies as well as a lack of
faithful and accessible animal models.
BIN1 is a scaffolding protein involved in the tubular
invagination of membranes and in membrane trafficking (
8 – 10
).
Alternative splicing of the BIN1 gene generates at least 12 isoforms
with diverse functions (
11 – 14
). All known protein isoforms
possess an N-terminal amphipathic helix, a BAR
(Bin/Amphiphysin/Rvs) domain able to sense and promote membrane
curvature through dimerization and a C-terminal SH3 domain
important for protein – protein interactions (
15 – 17
). Variability
arises in the protein’s central region, where a clathrin-binding
domain is present in neuronal isoforms and a PI-binding domain
is found almost exclusively in skeletal muscle isoforms
(
11,13,18
). Five of the six partial loss-of-function BIN1
mutations in CNM described to date are located in the
ubiquitously expressed and evolutionarily conserved BAR and SH3
domains (Fig. 1D). BAR missense mutations strongly decrease
BIN1 membrane tubulating properties in cultured cells,
whereas SH3 truncating mutations impair interactions with
DNM2 (7). Only recently was the first human BIN1 splice
mutation affecting the muscle-specific PI-binding domain identified,
and found to result in an unusually progressive form of the
myopathy (
19
). The molecular basis of the muscle specificity of
CNM2 remains largely unresolved.
BIN1 expression increases dramatically during muscle cell
growth and differentiation (
12,20
). In humans, the subcellular
localization of BIN1 protein shifts during skeletal muscle
development. BIN1 immunoreactivity appears as longitudinal
striations along the muscle fiber in (...truncated)