Regulation of LINC complex assembly and turnover in budding yeast

核膜是一个双层的细胞室，包裹着储存染色体的细胞核。它提供了一种将核质与细胞质分开的屏障。核膜上的小开口允许分子通过，但细胞质力量必须直接传递到核膜上，才能推动染色体运动和核迁移。这种力的传递是通过进化上保守的核骨架到细胞骨架(LINC)蛋白质复合体来实现的，它嵌入在核膜中。LINC复合体是如何形成和调节以在细胞内执行不同的核活动的，这两个问题都有待进一步阐明。本项目旨在剖析LINC复合体组装和维护的分子机制。Baker‘s酵母被用作遗传模型来确定LINC复合体组装的化学计量比和控制LINC蛋白质降解的信号。从酵母中获得的知识有望对理解LINC在动植物中的组装和功能具有普遍意义。该项目涉及培养细胞和分子生物学的本科生和研究生，包括少数民族学生。这也使佛罗里达州立大学每年开设的酵母遗传学(BSC 3402L)课程的不同学生群体受益，每年为约24名大学生提供进行目标导向研究的经验。典型的LINC复合体由一对完整的膜蛋白组成：太阳域蛋白定位于内核膜，Kash结构域蛋白定位于外核膜。哺乳动物编码5个不同的SUN基因和6个Kash同源物，而陆地植物拥有大量SUN和Kash样蛋白，可能形成不同的LINC复合体。酵母只有一种SUN蛋白，Mps3，以及两种类似Kash的蛋白，Mps2和Csm4。十多年来，人们一直推测Mps3与Mps2或Csm4配对，形成两个独立的异二聚体LINC络合物。初步研究意外地发现，Csm4、Mps2和Mps3在端粒形成了一个异三聚体LINC复合体。此外，LINC蛋白是在细胞周期中被降解的目标。假设靶向蛋白降解是调节LINC组装和功能的关键过程。为了验证这一假设，将实现三个特定的目标：(1)确定酵母LINC复合体形成的化学计量比；(2)确定LINC在核外围组装的空间调节；(3)确定LINC蛋白周转的时间调节。在第一个目标下，将确定酵母LINC复合体的相互作用蛋白结构域。在第二个目标下，将表征LINC复合体在端粒和中心体组装的特异性。在第三个目标下，将确定控制LINC蛋白周转的信号通路。这项拟议的研究有望在真核模型中揭示LINC组装和动态平衡的分子细节。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.