Regulation of LINC complex assembly and turnover in budding yeast

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The nuclear envelope, a double-membraned cellular compartment, encloses the nucleus where the chromosomes are stored. It provides a barrier that separates the nucleoplasm from the cytoplasm. Small openings on the nuclear envelope allow molecules to pass, but cytoplasmic forces must transmit directly across the nuclear envelope to power chromosome movement and nuclear migration. Relay of such forces is carried out by the evolutionarily conserved LInker of Nucleoskeleton to Cytoskeleton (LINC) protein complex, which is imbedded in the nuclear envelope. How the LINC complex is formed and modulated to carry out diverse nuclear activities inside the cell both remain to be further elucidated. This project aims to dissect the molecular mechanisms by which the LINC complex is assembled and maintained. Baker’s yeast is used as a genetic model to determine the stoichiometry of LINC complex assembly and the signal that controls LINC protein degradation. Knowledge gained from yeast is expected to have general implications for understanding LINC assembly and function in animals and plants. This project involves the training of undergraduate and graduate students, including minority students, in cell and molecular biology. It also benefits a diverse group of students who enroll in the Yeast Genetics (BSC 3402L) course taught annually at Florida State University, providing ~24 college students each year with experience in conducting goal-oriented research.The canonical LINC complex is composed of a pair of integral membrane proteins: the SUN-domain protein localized to the inner nuclear membrane, and the KASH-domain protein to the outer nuclear membrane. Mammals encode five different SUN genes and six KASH homologs, whereas land plants possess numerous SUN- and KASH-like proteins, potentially forming divergent LINC complexes. Yeast has only one SUN protein, Mps3, and two KASH-like proteins, Mps2 and Csm4. It has been speculated for more than a decade that Mps3 pairs with either Mps2 or Csm4 to form two independent heterodimeric LINC complexes. Preliminary studies revealed, unexpectedly, that Csm4, Mps2 and Mps3 form a heterotrimeric LINC complex at the telomere. Furthermore, LINC proteins are targeted for degradation during the cell cycle. The hypothesis is that targeted proteolysis is a key process that regulates LINC assembly and function. Three specific aims will be fulfilled to test this hypothesis: (1) determine the stoichiometry of yeast LINC complex formation, (2) determine the spatial regulation of LINC assembly at the nuclear periphery, and (3) determine the temporal regulation of LINC protein turnover. Under the first aim, the interacting protein domains of the yeast LINC complex will be determined. Under the second aim, the specificity of LINC complex assembly at the telomere and the centrosome will be characterized. Under the third aim, the signaling pathway that controls LINC protein turnover will be determined. The proposed research is expected to reveal the molecular details of LINC assembly and homeostasis in a eukaryotic model.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.