Exploration of the possible role(s) of two pore channel type 1 (TPC1)-mediated Ca²⁺ release during skeletal muscle development in zebrafish embryos

Abstract

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. I report that the endolysosomal ion channel, two-pore channel type 1 (TPC1), mediates localized, non-propagating Ca²⁺ transients that play a distinct and required role in the capture and attachment of skeletal muscle cells at the MJs. Use of antisense morpholinos or CRISPR/Cas9 mutagenesis to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including skeletal muscle cells detaching from or crossing the vertical myosepta. I also report that TPC1-decorated endolysosomes are dynamically associated with the MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or function disrupted endolysosomal trafficking and resulted in an abnormal distribution of β-dystroglycan (a key transmembrane component of the dystrophin-associated protein complex). Together, my data suggest that localized TPC1-generated Ca²⁺ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain the MJ following the onset of contractile activity. In a second series of experiments, I also investigated the possible roles of both TPC1 and two-pore channel type 2 (TPC2) during the formation of the zebrafish notochord, a specialized midline structure that serves structural and patterning roles during embryogenesis. As notochord vacuoles have been shown to be lysosome-related organelles, I hypothesized that TPCs might contribute to their biogenesis. My data suggest isoform specific roles for TPC1 and TPC2 in the development of the perinotochordal sheath, and the notochord vacuoles, respectively. Taken together, my results add new knowledge to how TPCs and endolysosomal-generated Ca²⁺ signaling contribute to the regulation of zebrafish myogenesis.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Andrew Leitch MILLER (Supervisor)