Caractérisation du sarcolemme dans les dystrophies musculaires des ceintures

Les dystrophies musculaires des ceintures (LGMD) sont un groupe hétérogène de dystrophies musculaires à progression lente. Des mutations du gène de la dysferline causent la LGMD de type 2B, mutations dans le gène de la cavéoline-3 (cav-3) causent LGMD de type 1C et des mutations dans le gène anoctamine-5 (ano-5) sont liées aux LGMD. Dans le but d'analyser les mécanismes moléculaires des LGMD et d'étudier les potentielles interactions de la dysferline, de la cav-3 et de l'ano5, des expériences sur des cellules musculaires primaires portant des mutations associées aux gènes DYSF, CAV3 et ANO5 ont été analysées. Les études d'immunomarquage ont montré que la protéine dysferline et la cav-3 sont partiellement colocalisées dans des structures vésiculaires de la membrane plasmique des myotubes primaires humains. La purification biochimique des "detergent-resistant membranes" issus des myotubes différenciés a montré que la dysferline est associée aux " lipid raft " liées aux cytosquelettes d'actine. L'analyse de la microscopie électronique sur les myotubes issus des muscles des patients atteints de LGMD a montré des altérations dans l'abondance des cavéoles à la membrane plasmique qui est en corrélation avec les mutations causant la maladie. L'analyse de l'ultrastructure cellulaire a montré que la dysferline est localisée à la membrane plasmique mais également dans des vésicules cytosoliques. L'immunopurification de ces vésicules contenant de la dysferline a révélé la présence d'environ 500 protéines détectées par LC-MS, ce qui pourrait représenter des protéines structurales vésiculaires, ainsi que des nouveaux partenaires potentiels d'interaction de la dysferline.Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of slowly progressive muscular dystrophies with common features such as hyperCKemia and skeletal muscle weakness. Mutations in the dysferlin gene cause LGMD 2B, in the caveolin-3 (cav-3) gene LGMD 1C and in the anoctamin-5 (ano-5) gene LGMD 2L, respectively. In order to reveal the molecular mechanisms underlying LGMD and to investigate the putative interactions of dysferlin, cav-3, and ano5, primary skeletal muscle cell lines with disease-related mutations in DYSF, CAV3, and ANO5 have been analyzed. Immunolabeling studies revealed that dysferlin and cav-3 are partially colocalized in vesicular structures at the plasma membrane. Biochemical purification of detergent-resistant membranes from differentiated myotubes showed that dysferlin is associated with lipid rafts linked to the actin-cytoskeleton. Transmission electron microscopy analysis of myotubes revealed alterations of caveolae abundance at the plasma membrane correlating with disease-causing mutations. Ultrastructural studies revealed localization of dysferlin at the plasma membrane, but also in cytosolic vesicles. These vesicles contained a subset of approximately 500 proteins detected by LC-MS, which might represent vesicular structural proteins, vesicle cargo, and putative new dysferlin interaction partners. Results from this study lead to the conclusion that caveolae play a crucial role in the context of LGMD. Dysferlin and cav-3 seem to be closely linked on structural as well as on functional level. Our results confirm that dysferlin is localized in cytosolic vesicles, which are involved in multiple cellular processes

The role of intraspinal sensory neurons in the control of quadrupedal locomotion

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Structural insights into the activation mechanism of dynamin-like EHD ATPases

Eps15 (epidermal growth factor receptor pathway substrate 15)homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50 degrees rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism

Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

<div><p>Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in <em>DYSF</em> lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.</p> </div

Dysferlin-peptides redirect mutant dysferlin to the sarcolemma in primary human myotubes.

<p>Primary human myotubes carrying dysferlin missense mutations were treated with the TAT-labeled dysferlin-peptides. Dysferlin was detected by anti-dysferlin ab. Nuclei are stained with Hoechst. Missense mutated dysferlin aggregates within the myotubes (<b>A, D</b>). After treating human myotubes with 10mer peptides (<b>B, E</b>) harboring the missense mutation mutant dysferlin can be localized at sarcolemmal sites whereas nonsense peptides (<b>C</b>) do not elicit this effect. (<b>A–C</b>) Primary human myotubes expressing <i>DYSF</i> p.G299R. Experiment performed 9x. (<b>A</b>) No peptides added. (<b>B</b>) Peptide A2 (10-mer) added corresponding to <i>DYSF</i> p.G299R. (<b>C</b>) Nonsense peptide added (control). (<b>D–E</b>) Primary human myotubes harboring the dysferlin mutation p.L1341P. Experiment performed 7x. (<b>D</b>) No peptides added. (<b>E</b>) Peptide B2 (10-mer) added corresponding to <i>DYSF</i> p.L1341P. (<b>F</b>) Sarcolemmal dysferlin localization in a normal human myotube. Bar: 10 µm. Arrows indicate reallocated dysferlin to sarcolemmal sites.</p

Specific peptides cause functional recovery in dysferlin-deficient myotubes: Interventional atomic force microscopy.

<p>Mechanical sarcolemmal wounding was induced by atomic force microscopy. In all experiments 2 µm longitudinal lesions were set. Arrows indicate the lesion site. The instrument shown is the cantilever used for membrane wounding. (<b>A–C</b>) Normal human myotube 5, 19 and 30 seconds after wounding. The lesion is hardly detectable and closes rapidly. (<b>D–F</b>) <i>DYSF</i> p.L1341P primary human myotube 6, 20 and 90 sec after wounding. The lesion continuously increases in size. (<b>G–I</b>) <i>DYSF</i> p.L1341P primary human myotube treated with corresponding B2 peptide 6, 22 and 30 seconds after wounding. The lesion disappears rapidly. Scale bar: 10 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049603#pone.0049603.s011" target="_blank">video S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049603#pone.0049603.s012" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049603#pone.0049603.s013" target="_blank">S7</a>.</p

TAT-labeled dysferlin-peptides in primary human myotubes localize to the ER.

<p>Peptide B2 corresponding to <i>DYSF</i> p.L1341P was labeled with ATTO-495-ME fluorescent dye (red) and added to <i>DYSF</i> p.L1341P human myotubes. (<b>A</b>) <i>DYSF</i> p.L1341P human myotube immediately after addition of ATTO-495-ME-peptide B2 to <i>DYSF</i> p.L1341P human myotubes, (<b>B</b>) after 8 minutes and (<b>C</b>) after 4 hours. (<b>D</b>) Immunostain using anti-calnexin ab (blue). Perfect co-localization of ATTO-495-ME-peptide B2 and ER marker calnexin. No dysferlin-peptide detected at the sarcolemma. Bar: 10 µm. See also video S8.</p

Dysferlin-derived peptides.

<p>Dysferlin-derived peptides.</p

Dysferlin-peptides redirect mutant dysferlin in transfected C2C12 cells.

<p>C2C12 cells were transfected with either GFP-tagged wildtype human dysferlin cDNA or missense-mutated dysferlin cDNA <i>DYSF</i> p.G299R or p.L1341P. Transfected cells were treated with TAT-labeled dysferlin-peptides corresponding to the mutation. (<b>A–C</b>) Wildtype human dysferlin-GFP tagged. (<b>A</b>) Dysferlin-GFP. (<b>B</b>) Localization of dysferlin is confirmed by immunostaining using an anti-dysferlin ab. (<b>C</b>) merge. (<b>D–F</b>) <i>DYSF</i> p.G299R-GFP tagged. Experiment performed 14×. (<b>D</b>) No peptides added. GFP-dysferlin is not expressed at the plasma membrane. (<b>E</b>) Peptide A2 (10-mer) added corresponding to <i>DYSF</i> p.G299R. GFP-dysferlin relocalizes to sarcolemmal sites. (<b>F</b>) Peptide A1 corresponding to WT dysferlin added. Distribution of dysferlin is granular but not expressed at the sarcolemma. (<b>G–I</b>) <i>DYSF</i> p.L1341P-GFP tagged. Experiment performed 12×. (<b>G</b>) No peptides added. (<b>H</b>) Peptide B2 (10-mer) corresponding to <i>DYSF</i> p.L1341P supports reallocation of dysferlin to the sarcolemma. (<b>I</b>) Peptide B4 (15-mer) corresponding to <i>DYSF</i> p.L1341P. 10mer peptides carrying the corresponding mutation were most effective. Bar: 10 µm. Inserts represent enlarged boxed areas.</p

TAT-labeled dysferlin-peptides reduce the expression of the ER stress sensor BiP.

<p>(<b>A</b>) In <i>DYSF</i> p.G299R human myotubes <i>HSPA5</i> gene expression is significantly up-regulated. Data represent median + SEM, n = 9/group. (<b>B and C</b>) Mutant peptides decrease <i>HSPA5</i> gene expression in <i>DYSF</i> p.G299R human myotubes most effectively (RT-PCR). Consequently, treatment with specific peptide A2 reduces BiP protein expression. C1: no peptides added; C2: addition of nonsense peptide; C3: treatment with specific peptide A2. The nonsense peptide serves as a control and its amino-acid sequence has no analogy to dysferlin. Data represent median + SEM, n = 3/group. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049603#pone.0049603.s005" target="_blank">Fig. S5</a> provides additional information on statistics.</p