Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy.

Centronuclear myopathies are characterized by muscle weakness and abnormal centralization of nuclei in muscle fibers not secondary to regeneration. The severe neonatal X-linked form (myotubular myopathy) is due to mutations in the phosphoinositide phosphatase myotubularin (MTM1), whereas mutations in dynamin 2 (DNM2) have been found in some autosomal dominant cases. By direct sequencing of functional candidate genes, we identified homozygous mutations in amphiphysin 2 (BIN1) in three families with autosomal recessive inheritance. Two missense mutations affecting the BAR (Bin1/amphiphysin/RVS167) domain disrupt its membrane tubulation properties in transfected cells, and a partial truncation of the C-terminal SH3 domain abrogates the interaction with DNM2 and its recruitment to the membrane tubules. Our results suggest that mutations in BIN1 cause centronuclear myopathy by interfering with remodeling of T tubules and/or endocytic membranes, and that the functional interaction between BIN1 and DNM2 is necessary for normal muscle function and positioning of nuclei.

The myotubularin-amphiphysin 2 complex in membrane tubulation and centronuclear myopathies.

Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1-mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.

Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy.

The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

Mild functional differences of dynamin 2 mutations associated to centronuclear myopathy and Charcot-Marie Tooth peripheral neuropathy.

The large GTPase dynamin 2 is a key player in membrane and cytoskeletal dynamics mutated in centronuclear myopathy (CNM) and Charcot-Marie Tooth (CMT) neuropathy, two discrete dominant neuromuscular disorders affecting skeletal muscle and peripheral nerves respectively. The molecular basis for the tissue-specific phenotypes observed and the physiopathological mechanisms linked to dynamin 2 mutations are not well established. In this study, we have analyzed the impact of CNM and CMT implicated dynamin 2 mutants using ectopic expression of four CNM and two CMT mutations, and patient fibroblasts harboring two dynamin 2 CNM mutations in established cellular processes of dynamin 2 action. Wild type and CMT mutants were seen in association with microtubules whereas CNM mutants lacked microtubules association and did not disrupt interphase microtubules dynamics. Most dynamin 2 mutants partially decreased clathrin-mediated endocytosis when ectopically expressed in cultured cells; however, experiments in patient fibroblasts suggested that endocytosis is overall not defective. Furthermore, CNM mutants were seen in association with enlarged clathrin stained structures whereas the CMT mutant constructs were associated with clathrin structures that appeared clustered, similar to the structures observed in Dnm1 and Dnm2 double knock-out cells. Other roles of dynamin 2 including its interaction with BIN1 (amphiphysin 2), and its function in Golgi maintenance and centrosome cohesion were not significantly altered. Taken together, these mild functional defects are suggestive of differences between CMT and CNM disease-causing dynamin 2 mutants and suggest that a slight impairment in clathrin-mediated pathways may accumulate over time to foster the respective human diseases.

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.

BACKGROUND: In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). METHODOLOGY: To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. CONCLUSION: Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM.

Novel molecular diagnostic approaches for X-linked centronuclear (myotubular) myopathy reveal intronic mutations.

X-linked centronuclear myopathy (XLMTM), also called myotubular myopathy, is a severe congenital myopathy characterized by generalized hypotonia and weakness at birth and the typical histological finding of centralization of myo-nuclei. It is caused by mutations in the MTM1 gene encoding the 3-phosphoinositides phosphatase myotubularin. Mutations in dynamin 2 and amphiphysin 2 genes lead to autosomal forms of centronuclear myopathy (CNM). While XLMTM is the most frequent and severe form of CNM, no mutations are found in about 30% of patients by sequencing all MTM1 exons. Moreover, the impact of MTM1 sequence variants is sometimes difficult to assess. It is thus important to devise a complete molecular diagnostic strategy that includes analysis of the myotubularin transcript and protein expression. We therefore developed novel antibodies against human myotubularin and showed that they are able to detect the endogenous protein by direct Western blot from muscle samples and from cultured cells. In conjunction with RT-PCR analysis we validated the consequences of missense and splice mutations on transcript integrity and protein level. We also detected and characterized a novel deep intronic mutation consisting of a single nucleotide change that induces exonisation of a conserved intronic sequence. Patients with centronuclear myopathy and no molecular diagnosis should be investigated for MTM1 defects at the cDNA and protein level.

A novel PtdIns3P and PtdIns(3,5)P2 phosphatase with an inactivating variant in centronuclear myopathy.

In eukaryotic cells, phosphoinositides are lipid second messengers important for many cellular processes and have been found dysregulated in several human diseases. X-linked myotubular (centronuclear) myopathy is a severe congenital myopathy caused by mutations in a phosphatidylinositol 3-phosphate (PtdIns3P) phosphatase called myotubularin, and mutations in dominant centronuclear myopathy (CNM) cases were identified in the dynamin 2 gene. The genes mutated in autosomal recessive cases of CNMs have not been found. We have identified a novel phosphoinositide phosphatase (hJUMPY) conserved through evolution, which dephosphorylates the same substrates as myotubularin, PtdIns3P and PtdIns(3,5)P(2), in vitro and ex vivo. We found, in sporadic cases of CNMs, two missense variants that affect the enzymatic function. One of these appeared de novo in a patient also carrying a de novo mutation in the dynamin 2 gene. The other missense (R336Q) found in another patient changes the catalytic arginine residue of the core phosphatase signature present in protein tyrosine/dual-specificity phosphatases and in phosphoinositide phosphatases and drastically reduces the enzymatic activity both in vitro and in transfected cells. The inheritance of the phenotype with regard to this variant is still unclear and could be either recessive with an undetected second allele or digenic. We propose that impairment of hJUMPY function is implicated in some cases of autosomal CNM and that hJUMPY cooperates with myotubularin to regulate the level of phosphoinositides in skeletal muscle.