Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Golgi Complex Organization in Skeletal Muscle: A Role for Golgi‐Mediated Glycosylation in Muscular Dystrophies?

The Golgi complex (GC) is the central organelle of the classical secretory pathway, and it receives, modifies and packages proteins and lipids en route to their intracellular or extracellular destinations. Recent studies of congenital muscular dystrophies in skeletal muscle suggest an exciting new role for an old and well-established function of the GC: glycosylation. Glycosylation is the exquisitely regulated enzymatic addition of nucleotide sugars to proteins and lipids mediated by glycosyltransferases (GTs). Mutations in putative Golgi-resident GTs, fukutin, fukutin-related protein and large1 cause these progressive muscle-wasting diseases. The appropriate localization of GTs to specific subcompartments of the Golgi is critical for the correct assembly line-like addition of glycan groups to proteins and lipids as they pass through the GC. Consequently, these studies of congenital muscular dystrophies have focused attention on the organization and function of the GC in skeletal muscle. In contrast to other cells and tissues, the GC in skeletal muscle has received relatively little attention; however, in recent years, several studies have shown that GC distribution in muscle is highly dynamic or plastic and adopts different distributions in muscle cells undergoing myogenesis, denervation, regeneration and maturation. Here, we review the current understanding of the dynamic regulation of GC organization in skeletal muscle and focus on the targeting of fukutin, fukutin-related protein and large1 to the GC in muscle cells.     

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.