Niche-mediated LINC Complex Regulation of the Muscle Stem Cell Transcriptome

Nissar, Aliyah A. 1 ; Davoudi, Sadegh 1 ; Montgomery-Song, Aaryn 1 ; Kim, Taehyung 1 ; Gomes, Edgar 2 ; Zhang, Zhaolei 1 ; Hodzic, Didier 3 ; Gilbert, Penney M. 1

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Instituto de Medicina Molecular, Universidade de Lisboa; 3. Dept of Ophthalmology & Vision, Washington University School of Medicine

A fundamental contributor of adult skeletal muscle regeneration is their resident stem cell population, termed satellite cells. Determining the mechanisms which regulate satellite cell fate will improve their therapeutic potential.

KASH and SUN are integral membrane protein domains that localize to the outer- and inner nuclear membranes, respectively. The domains interact to form a bridge across the nuclear membranes that link the cytoskeleton to the nuclear lamina lending to their designation as the LINC complex (Linker of Nucleoskeleton and Cytoskeleton). Importantly, disruption of this complex gives rise to various diseases including Emery-Dreifuss muscular dystrophy (EDMD).

Nesprins 1 and 2 possess KASH domains and can interact with cytoplasmic f-actin. Observations indicating that the Nesprin/Sun bridge can move chromosomes by transferring forces initiated outside the nucleus led to speculations that the complex might physically open inaccessible regions of the genome to modify gene expression. This lingering hypothesis is poised to address a cellular mechanotransduction quandary. Stem cells alter their fate in response to niche physical cues like tissue stiffness. However, mechanisms mediating transduction of external physical cues to internal gene expression are only now emerging. My thesis studies aim to fill these knowledge gaps by showing that forces generated at the cell surface lead to mechanical tension across the Nesprin/Sun complex to loosen regions of the chromatin that permit transcription of genes required for muscle stem cell specification. We hypothesize that the extracellular niche plays a role in regulating muscle stem cell self-renewal and specification through mechanotransduction signals propagated by the LINC complex.

We find that Nesprins, but not SUN proteins, are expressed in quiescent satellite cells using immunofluorescence. SUN expression is induced shortly (2 hrs) after culture suggesting that the Nesprin/Sun bridge is assembled during satellite cell activation. Knock down of Nesprin expression in satellite cells using siRNA showed an impairment of satellite cell proliferation. This impairment was also observed in myoblasts. Using a Pax7cre/Kash2 transgenic mouse model, where a dominant negative Nesprin disrupts the LINC complex in Pax7+ satellite cells upon tamoxifen administration, we see an impairment in muscle regeneration after a serial injury. Recently, it has been shown in literature that LINC complex regulates genome transcription in response to substrate rigidity. To uncover novel genes influenced by the transmission of extracellular mechanical stimuli, we performed RNAseq on primary myoblasts cultured upon soft or stiff substrates and expressing GFP or a Nesprin dominant-negative mutant that cannot bind f-actin. Our study hopes to provide new insight into the pathogenesis of Emery-Dreifuss muscular dystrophy, and elucidate mechanisms that regulate satellite cell fate.