Revealing the Role of IGFN1 in Skeletal Muscle

The role of the skeletal muscle protein Immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1) has proved elusive. There are several IGFN1 isoforms, none of which contain catalytic domains. Each isoform has a domain composition of immunoglobulin and fibronectin domains, suggesting a structural role in the sarcomere. IGFN1 was first discovered as an interacting partner of the disease-associated protein KY, and the current literature implicates IGFN1 in both atrophy and myoblast fusion. Here, characterisation of fusion and differentiation indexes. compared to wildtype, of a CRISPR/Cas9-generated, C2C12-derived, IGFN1 knockout cell line revealed fusion and differentiation defects. Furthermore, these cells display increased globular to filamentous actin ratios, indicating decreased actin polymerisation which potentially underlies the fusion defects observed. Crucially, the above phenotypes are ameliorated through expression of the IGF1_V1 isoform. Next, to identify IGFN1 interaction partners, IGFN1 fragments were purified and pull-down analysis was performed revealing the actin nucleator COBL as a potential interacting partner. This interaction subsequently validated through immunoprecipitation and colocalization. The role of COBL in myoblast fusion was investigated through overexpression experiments and the generation of a C2C12-derived knockout cell line. Initial characterisation points towards a role for COBL in myoblast fusion. Taken together, it is possible that IGFN1 influences actin remodelling, and therefore myoblast fusion, through its interaction with COBL

Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL

The Igfn1 gene produces multiple proteins by alternative splicing predominantly expressed in skeletal muscle. Igfn1 deficient clones derived from C2C12 myoblasts show reduced fusion index and morphological differences compared to control myotubes. Here, we first show that G:F actin ratios are significantly higher in differentiating IGFN1-deficient C2C12 myoblasts, suggesting that fusion and differentiation defects are underpinned by deficient actin remodelling. We obtained pull-downs from skeletal muscle with IGFN1 fragments and applied a proteomics approach. The proteomic composition of IGFN1 complexes identified the cytoskeleton and an association with the proteasome as the main networks. The actin nucleating protein COBL was selected for further validation. COBL is expressed in C2C12 myoblasts from the first stages of myoblast fusion but not in proliferating cells. COBL is also expressed in adult muscle and, as IGFN1, localizes to the Z-disc. We show that IGFN1 interacts, stabilizes and colocalizes with COBL and prevents the ability of COBL to form actin ruffles in COS7 cells. COBL loss of function C2C12-derived clones are able to fuse, therefore indicating that COBL or the IGFN1/COBL interaction are not essential for myoblast fusion

IGFN1_v1 is required for myoblast fusion and differentiation.

Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres

Using information entropy to optimise and communicate certainty of continental scale tectonic models

&amp;lt;p&amp;gt;Antarctic subglacial properties impact geothermal heat, subglacial sedimentation, and glacial isostatic adjustment; critical parameters for predicting the ice sheet&amp;#039;s response to warming oceans. However, the tectonic architecture of the Antarctic interior is unresolved, with results dependent on datasets or extrapolation used. Most existing deterministic suggestions are derived from qualitative observations and often presented as robust results; however, they hide possible alternative interpretations.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;amp;#160;&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;Using information entropy as a measure of certainty, we present a robust tectonic segmentation model generated from similarity analysis of multiple geophysical and geological datasets. The use of information entropy provides us with an unbiased and transparent metric to communicate the ambiguities from the uncertainties of qualitative classifications. Information theory also allows us to test and optimise the methods and data to evaluate how choices impact the distribution of alternative output maps. We further discuss how this metric can quantify the predictive power of parameters as a function of regions with different tectonic settings.&amp;lt;/p&amp;gt;</jats:p