Mitotic Roles for Cytoplasmic Dynein and Implications for Brain Developmental Disease: a Dissertation

Abstract

Cytoplasmic dynein has been implicated in a wide range of functions. Originally characterized as being responsible for retrograde axonal transport, its has also been shown to traffic vesicular organelles (Golgi, endosome and lysosome distribution), transport viral particles to the nucleus, and participate in microtubule organization. During mitosis, cytoplasmic dynein is thought to function in spindle pole focusing and prometaphase kinetochore capture. This thesis explores the mitotic roles of cytoplasmic dynein. The first chapter addresses the role of cytoplasmic dynein in kinetochore activity. Using immunofluoresence microscopy, a number of motors and related proteins were observed at the primary, but not secondary, constrictions of prometaphase multicentric chromosomes. The proteins assessed included the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1, and p150Glued), the kinesin related proteins CENP-E and MCAK, and the proposed structural and checkpoint proteins CENP-F, HZW10, and MAD2. The differential localization of these proteins offered new insight into the assembly and composition of both active and inactive centromeres, and provided a molecular basis for the apparent inactivity of the latter during chromosome segregation. The second chapter characterizes LIS1, a protein that is defective in the developmental brain disease type1 lissencephaly. Mutations in the LIS1 gene cause gross histological disorganization of the developing cerebral cortex resulting in a nearly smooth brain surface. Because genetic evidence from lower eukaryotes suggested that LIS1 acted within the cytoplasmic dynein pathway, it was of interest to analyze LIS1 in terms of cytoplasmic dynein function. LIS1 was found to coimmunoprecipitate with cytoplasmic dynein and its companion complex dynactin. During mitosis LIS1 localized to the prometaphase kinetochore, spindle microtubules and the cell cortex, known sites for cytoplasmic dynein binding. Interference with endogenous LIS1 in cultured mammalian cells displaced dynein localization and disrupted mitotic progression. LIS1 was concluded to participate in cytoplasmic dynein functions, but only during mitosis. Data presented in the final chapter further characterizes LIS1's interactions with microtubules, cytoplasmic dynein and the mammalian homologue of NUDC. LIS1 was not found to co-fractionate with microtubules, nor did overexpression of LIS1 cause visible effects on microtubule organization or dynamics. GFP-LIS1 was shown to ride along the plus ends of growing microtubules. Though LIS1 was not found to have a direct effect on microtubules, it may regulate dynein's microtubule binding activity. LIS1 was found to co-immunoprecipitate with a co-overexpressed cytoplasmic dynein subunit substantiating the existence of a dynein LIS1 supercomplex. Furthermore, association of these proteins increased markedly in mitotically blocked samples. LIS1's regulation of cytoplasmic dynein may change the capacity of the motor to efficiently manipulate its mitotic cargoes, dramatically effecting the timing of cell division. This disruption has implications for the fundamental role of cytoplasmic dynein during early embryonic development.