Characterization of Hemicentin in C. elegans
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2020-04-21Author
Ahumada, Abraham
Haworth, Emma
Ross, Kylie
Sowinski, Halee
Antony, Keerthi
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Characterization of Hemicentin in C. elegans
Emma Haworth, Kylie Ross, Halee Sowinski, Keerthi Antony, Abraham Ahumada
Department of Biology, Baylor University, One Bear Place 97388, Waco, TX 76798, U.S.A
In the nematode, Caenorhabditis elegans, the him-4 gene linked to the X chromosome
encodes hemicentin protein, a component of the extracellular matrix (ECM), which is
characterized by 45 immunoglobulin repeats, and fibulin-like domains. The ECM is a
three-dimensional network composed of proteins and sugars deposited outside of the cell. ECM
proteins are typically large, glycosylated, and contain repeats and motifs for cell binding.
Hemicentin is specifically present in the basement membrane (BM), a special sheet-like ECM,
that plays an important role in cell migration and tissue attachment, and stability of mitotic germ
cells. HIM-4 contains six RGD motifs, a protein sequence specific to the integrin binding
receptor. In the following study, CRISPR gene editing was used to create mutations in him-4 at
two of the six RGD sequences. These sequences were targeted to replace the D amino acid
(Aspartic Acid) with the E amino acid (Glutamic Acid). This mutation in him-4 causes defective
phenotypes related to cell binding. We have isolated several targeted animals with tissue
fragility, suggesting that the RGD sequence is vital for the function of the protein; the gene
editing may interfere with hemicentin binding to the integrin receptor. The disruption of the ECM
causes improper attachment of the gonad BM to epithelial BM leading to the hemorrhaging of
the gonads and the intestines in C. elegans. The observation of the hemorrhaging phenotype
and the single-worm PCR will be used to detect CRISPR-induced homozygous alleles. This
research may allow for further studies on gonad development and human orthologs of the
hemicentin protein. The connection between the hemicentin protein and the ECM deformities
may offer insight into diseases associated with tissue fragility.