The cell biology field has outstanding working knowledge of the fundamentals of membrane-trafficking pathways, which are of critical importance in health and disease. Current challenges include understanding how trafficking pathways are fine-tuned for specialized tissue functions and during development. In parallel, the ENCODE project and numerous genetic studies have revealed that alternative splicing regulates gene expression in tissues and throughout development at a post-transcriptional level. This Review summarizes recent discoveries demonstrating that alternative splicing affects tissue specialization and membrane-trafficking proteins during development, and examines how this regulation is altered in human disease. We first discuss how alternative splicing of clathrin, SNAREs and BAR-domain proteins influences endocytosis, secretion and membrane dynamics, respectively. We then focus on the role of RNA-binding proteins in the regulation of splicing of membrane-trafficking proteins in health and disease. Overall, our aim is to comprehensively summarize how trafficking is molecularly influenced by alternative splicing and identify future directions centered on its physiological relevance.

Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.

The long-term goal of the proposed research is to characterize the function of the motif (''M-domain'') of cardiac myosin binding protein C (cMyBP-C) in damping spontaneous oscillatory contractions (SPOC) in skinned cardiomyocytes. The motif aids in binding of cMyBP-C to both myosin and actin, and changes to its phosphorylation affect cMyBP-C affinity for both. Reduced phosphorylation of the motif is common in many cases of hypertrophic cardiomyopathy (HCM), and cMyBP-C mutations that reduce cMyBP-C expression are present in approximately 50% of cases of HCM. Recently, we developed a new ''cut-and-paste'' method that allows sequential removal of domains C0-C7 (including the M-domain) and covalent replacement with any desired recombinant protein containing SpyCatcher (SC). Using this new method we found that acute removal of cMyBP-C domains C0-C7 resulted in the appearance of SPOC in Ca 2þ activated skinned cardiomyocytes, whereas covalent replacement with recombinant C0-C7-SC domains damped SPOC. Here we tested whether the motif is necessary to damp SPOC when C0-C7-SC is added back by comparing C0-C7-SC that contains motif and C0-C7DM-SC that lacks the motif. Results showed that C0-C7DM-SC did not damp SPOC, implicating the motif as a critical domain in regulating the process of SPOC. Ongoing studies will investigate additional cMyBP-C recombinant protein constructs to determine whether the motif alone is sufficient to damp SPOC and whether phosphorylation of the motif affects cMyBP-C's capability to damp SPOC. This work is supported by NIH HL080367 and HL140925 and Interdisciplinary Training in Cardiovascular Research T32HL007249.

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The reprogramming of alternative splicing patterns during development is a hallmark of tissue maturation and identity. Heart and skeletal (striated) muscle tissues exhibit the most highly conserved splicing programs, which substantially change during postnatal development. Notably, alterations of these splicing events are associated with severe cardiac and muscular diseases. In striated muscle, a collection of genes encoding membrane trafficking proteins are spliced in a developmental stage‐specific manner. However, few splice isoforms have been characterized, and little is known about their functional roles during heart and skeletal muscle development. We hypothesize that coordinated splicing of membrane trafficking genes represents a splicing network, which functions to regulate the transport of cargo that is critical for muscle development and homeostasis. Here, we characterize three proteins involved in vesicle‐mediated transport that are alternatively spliced in striated muscle: SNAP23, TMED2, and TRIP10. The synaptosome‐associated protein 23 (SNAP23) is a SNARE protein that mediates vesicle fusion with the plasma membrane during exocytosis, the transmembrane emp23 domain‐containing protein 2 (TMED2) regulates vesicle budding during secretion, and the thyroid hormone receptor interactor 10 (TRIP10) is involved in endocytosis and membrane tubulation. First, we found that during striated muscle development, the Snap23, Tmed2, and Trip10 pre‐mRNAs undergo splicing changes of a single exon. Second, these three splicing transitions are characterized by a shift from short to long isoform expression that are regulated by the RBPs quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1). Third, depletion of SNAP23 in muscle cells results in reduced cell viability and differentiation, which can be rescued with conditioned media from differentiated cells or by seeding cells on collagen‐coated plates. Finally, exclusive expression of the short SNAP23 isoform leads to increased fusion of myoblasts into myotubes that exhibit greater myotube area and a higher nuclei‐per‐myotube ratio. Taken together, our data suggest that splicing of Snap23 is an important regulator of myogenesis, where SNAP23 isoform expression controls secretion of cargo that is necessary for muscle cell fusion and myotube size. Preliminary studies show that disruption of TMED2 and TRIP10 isoform levels in muscle cells likewise affects differentiation. Elucidating the mechanisms of regulation and the functional consequences of splicing networks will be fundamental to our understanding of muscle development and disease.