Bruno Rinaldi

Phosphatase-dead myotubularin ameliorates X-linked centronuclear myopathy phenotypes in mice.

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1C375S and MTM1S376N) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1C375S mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.

Study of the Plant COPII Vesicle Coat Subunits by Functional Complementation of Yeast Saccharomyces cerevisiae Mutants

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23–Sec24 heterodimer following the subsequent recruitment of the Sec13–Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott–Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.

Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

Sorting of multivesicular bodies requires the endosomal-sorting complex required for transport (ESCRT) machinery. The kinases Pkh1/2 phosphorylate the ESCRT-0 subunit Vps27 on residue S613. Furthermore, this phosphorylation regulates ESCRT-I recruitment to endosomes.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy

Significance We present an identification of the relevance of WD40-repeat (WDR) genes in brain connectivity, highlighting the power of unbiased mouse studies in the field of neuroscience. We focus on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. WDR47 plays a role in progenitor proliferation, neuronal migration, and fiber tract projections in a similar fashion to LIS1 but with the distinctive particularity that WDR47 inhibits autophagic flux. This provides a functional link between autophagy biology and the C-terminal to LisH domain in mammals. Importantly, WDR47 uncovers an aspect of corpus callosum biology pointing toward a link between the regulation of microtubule dynamics and autophagic flux for axonal outgrowth and guidance. The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.

Expression of the neuropathy-associated MTMR2 gene rescues MTM1-associated myopathy

Myotubularins (MTMs) are active or dead phosphoinositides phosphatases defining a large protein family conserved through evolution and implicated in different neuromuscular diseases. Loss-of-function mutations in MTM1 cause the severe congenital myopathy called myotubular myopathy (or X-linked centronuclear myopathy) while mutations in the MTM1-related protein MTMR2 cause a recessive Charcot-Marie-Tooth peripheral neuropathy. Here we aimed to determine the functional specificity and redundancy of MTM1 and MTMR2, and to assess their abilities to compensate for a potential therapeutic strategy. Using molecular investigations and heterologous expression of human MTMs in yeast cells and in Mtm1 knockout mice, we characterized several naturally occurring MTMR2 isoforms with different activities. We identified the N-terminal domain as responsible for functional differences between MTM1 and MTMR2. An N-terminal extension observed in MTMR2 is absent in MTM1, and only the short MTMR2 isoform lacking this N-terminal extension behaved similarly to MTM1 in yeast and mice. Moreover, adeno-associated virus-mediated exogenous expression of several MTMR2 isoforms ameliorates the myopathic phenotype owing to MTM1 loss, with increased muscle force, reduced myofiber atrophy, and reduction of the intracellular disorganization hallmarks associated with myotubular myopathy. Noteworthy, the short MTMR2 isoform provided a better rescue when compared with the long MTMR2 isoform. In conclusion, these results point to the molecular basis for MTMs functional specificity. They also provide the proof-of-concept that expression of the neuropathy-associated MTMR2 gene improves the MTM1-associated myopathy, thus identifying MTMR2 as a novel therapeutic target for myotubular myopathy.

Amphiphysin (BIN1) negatively regulates dynamin 2 for normal muscle maturation

Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1–/– Dnm2+/– mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1–/– mice die perinatally from a skeletal muscle defect, Bin1–/– Dnm2+/– mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.

Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins.

ABSTRACT Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein–protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3&Dgr; mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2.

Cex1 is a new component of the COPI Golgi-to-vacuole intracellular trafficking machinery

During translational elongation, aminoacylated tRNA are supplied to the ribosome by RNA-interacting proteins. Cex1, a HEAT-containing protein, has been shown to participate to this tRNA channeling by interacting with aminoacylated tRNA during their export from the nucleus. Here, we show that Cex1 is a component of COPI (coatomer complex I) coated vesicles involved in the Golgi-to-vacuole trafficking pathway in Saccharomyces cerevisiae. Cex1 interacts with key components of the COPI coat Sec27, Sec28 and Sec33 proteins. Moreover, fluorescent microscopy indicates that Cex1 is not localized at the nuclear periphery as expected for an effector of nuclear tRNA channeling, but is observed on endosomal trans-Golgi network (TGN) positive structures. This localization relies on the vacuolar protein sorting receptor Vps10. Our data not only resolve the functional ambiguity regarding Cex1 homologues across species, but also point to the possibility to develop yeast-based models to study neurodegenerative disorders linked to the Human Cex1 homologue SCYL1.

C.P.10 Phosphatase inactive myotubularin rescues X-linked centronuclear (myotubular) myopathy phenotypes in mice

Abstract The X-linked centronuclear myopathy, also called myotubular myopathy, is a muscle disorder characterized by neonatal hypotonia and abnormal organelles positioning in skeletal muscle. This myopathy is due to different mutations in the MTM1 gene encoding the phosphoinositide phosphatase myotubularin. Disease-causing mutations are found all along the protein sequence and not only in the phosphatase catalytic domain. We investigated the link between myotubularin phosphatase activity and disease phenotypes. We used brewer yeast as a simple cellular model to analyze the in vivo phosphatase activity of different disease mutants. Our results show that mutations responsible for severe forms of myopathy are either active or inactive phosphatases. To further question this finding, we used the murine myotubularin knock-out model that reproduces faithfully the histopathological findings from human patients. Expression of phosphatase-inactive mutants improves the phenotypes of the knock-out mice comparable to wild-type myotubularin. This shows that the maintenance of normal skeletal muscles is largely independent from myotubularin phosphatase activity. Moreover, it could have important implications in the design of therapeutic approaches aiming at manipulating the phosphoinositide level in the different diseases linked to myotubularin homologues. Finally, this work underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.