The Requirement of Beta-Dystroglycan and a Sun1 Protein Interactome in Myonuclei

The nuclear lamina (NL) is composed of A- and B-type lamins, which assemble into a complex, insoluble meshwork beneath the inner nuclear membrane (INM). The lamina is anchored to the cytoskeleton by the LINC (Linker of the nucleoskeleton and cytoskeleton) complex which spans the NE and comprises INM SUN-domain proteins which bind their outer nuclear membrane (ONM) counterparts, KASH-domain proteins, in the perinuclear space. The NL and LINC complex, together with other NE proteins, have diverse functions in genome tethering, transcriptional regulation and nuclear positioning. -dystroglycan is an established cell adhesion protein which is thought to also function in the nucleus to stabilise a number of structures including the NL, but mechanisms behind this are elusive. It is hypothesised that -dystroglycan exerts its influence on the nucleus through the LINC complex, since previous mass spectrometry analyses found that Sun1 co-precipitated with -dystroglycan. A Sun1--dystroglycan interaction was interrogated using several techniques, with little evidence being found to support its existence in human myoblasts. Moreover, CRISPR-mediated ablation of dystroglycan in human myoblasts appears to not disturb nuclei as previously reported. -dystroglycan is also extensively modified post-translationally and proteolytic fragments are persistent within cells. Whether distinct -dystroglycan species have differential functions is unknown. Epitope-tag detection of fragments in situ revealed that proteolytic fragments become separately organised, furthering the notion that -dystroglycan is intricately controlled within the cell.
Sun1 is crucial during development as well as being implicated in muscle-wasting disorders both as a causative and disease modifying component. Lmna, encoding lamins A and C, is widely expressed in somatic cells, yet mutations give rise to a range of tissue-specific diseases, including muscular dystrophies, for reasons not fully understood. Ablation of Sun1 in Lmna mutant mice suppresses many of the Lmna-associated disease phenotypes revealing a potential functional interaction between the LINC complex and NL. It is postulated that nucleoplasmic protein interactions of Sun1 are altered in a tissue-specific manner, in response to the specific Lmna mutation present, that then elicit disease phenotypes. To identify potential nucleoplasmic Sun1 interacting proteins, the proximity dependent biotinylation 2C-BioID technique was used in myoblasts. 2C-BioID identified Lamin A and 6 novel Sun1 interactors which were verified using bimolecular fluorescence complementation. One of these novel Sun1 interactors was Ppm1a, so further implicate the NE and the LINC complex in the regulation of TGF signalling.