Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.

In skeletal muscle, the triad is a structure comprising a transverse (T)-tubule and sarcoplasmic reticulum (SR) cisternae. Triads constitute the basis of excitation-contraction coupling as the cradle of the Ca 2+ release complex. We have shown previously that triadin, a member of this complex, has shaping properties on reticulum membrane and is indirectly involved in a link between triads and microtubules. We have identified here that CLIMP-63 (also known as CKAP4), as the partner of triadin, is responsible for this association of triads and microtubules. Triadin and CLIMP-63 interact through their respective luminal domains and the shaping properties of triadin depend on the capacity of CLIMP-63 to bind microtubules with its cytosolic portion. In skeletal muscle, CLIMP-63 is localized in the SR, including triads, and is associated with the Ca 2+ release complex through its interaction with triadin. Knockout of triadin in muscles results in the delocalization of CLIMP-63 from triads, its dissociation from the Ca 2+ release complex and a disorganization of the microtubule network. Our results suggest that the association of triadin and CLIMP-63 could be involved in the shaping of SR terminal cisternae and in the guidance of microtubules close to the triads.

SummaryThe terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad.