Final Report Summary - DYNEINOME (Cytoplasmic Dynein: Mechanisms of Regulation and Novel Interactors)
Animal cells rely on molecular motors to distribute intracellular components and organize their cytoplasmic content. The motor complex cytoplasmic dynein 1 (dynein) uses microtubules as tracks and participates in a wide range of cellular activities, which include the transport and positioning of organelles and the segregation of chromosomes during cell division. Dynein consists of multiple subunits and associates with a host of co-factors in a context-specific manner to form a megadalton-sized transport machine. Mutations in dynein and its co-factors have been linked to a variety of nervous system disorders. Elucidating the molecular basis of how dynein is regulated will help us understand the functional diversity of the motor and its pathogenic mis-regulation in disease. In this project, we used live-cell fluorescence microscopy, genome editing, and biochemical approaches in the roundworm Caenorhabditis elegans and human cultured cells to study the cellular roles and molecular mechanisms of conserved dynein co-factors that regulate localization and activity of the motor.
First, we asked how dynein is recruited to the kinetochore, a multi-protein assembly on chromosomes that interacts with spindle microtubules to segregate chromosomes during cell division. We identified and characterized specific protein-protein interactions through which the adaptor protein Spindly recruits dynein and its activator dynactin. This analysis revealed that similar protein-protein interactions are also used by other adaptor proteins that recruit dynein to organelles. The results support the view that there is considerable overall conservation in the mechanism of dynein recruitment to diverse cargo, despite the fact that many of the adaptor proteins involved do not share obvious common ancestry. We also characterized how Spindly interacts with its receptor at the kinetochore, the Rod-Zw10-Zwilch complex (RZZ). We found that RZZ and Spindly cooperate to form an outermost expanded kinetochore domain during cell division in human cultured cells, most likely by self-assembling into filamentous oligomers. Expansion of the kinetochore surface in early mitosis facilitates the capture of astral microtubules by dynein and other microtubule binding proteins present at the kinetochore, thereby accelerating the process of spindle assembly. Once kinetochores become attached to microtubules, dynein transports RZZ-Spindly and other outermost kinetochore components towards spindle poles as part of a motor-cargo complex, which contributes to the disassembly of the expanded kinetochore domain. This leaves a compact and microtubule-attached core kinetochore in place for the segregation of chromosomes to daughter cells. Our results, along with concurrent studies from other groups, define a new role for the RZZ-Spindly-dynein module as a key regulator of adaptive kinetochore size that enhances efficiency and accuracy of spindle assembly.
Second, we asked how the different subunits of the mega-dalton dynactin complex contribute to the function of this essential dynein co-factor. In the dividing C. elegans early embryo, we found that dynactin's microtubule binding activity, which resides in its largest subunit p150, helps dynein pull on microtubules to position the microtubule organizing center, the centrosome. We also discovered that the peripheral dynactin subunit p27 is required for targeting dynein-dynactin to kinetochores but not to other subcellular sites. This shows that while dynactin is a general activator of dynein motility, some of its subunits participate in specific cellular processes.
Third, we sought to identify new dynein pathway genes in a genome-wide RNAi screen in C. elegans. We used animals in which dynein function is partially compromised because the gene coding for one its co-factors, NudE, is lacking. We looked for genes whose downregulation of expression made NudE knockout animals more sick than control animals. This enhancer screen identified several dozen genes, many of which have functions in cell division. Characterization of two of the genes showed how their corresponding proteins co-operate with NudE at the kinetochore to ensure faithful chromosome segregation. How other genes identified in the screen relate to the dynein pathway will be interesting to investigate in the future.
First, we asked how dynein is recruited to the kinetochore, a multi-protein assembly on chromosomes that interacts with spindle microtubules to segregate chromosomes during cell division. We identified and characterized specific protein-protein interactions through which the adaptor protein Spindly recruits dynein and its activator dynactin. This analysis revealed that similar protein-protein interactions are also used by other adaptor proteins that recruit dynein to organelles. The results support the view that there is considerable overall conservation in the mechanism of dynein recruitment to diverse cargo, despite the fact that many of the adaptor proteins involved do not share obvious common ancestry. We also characterized how Spindly interacts with its receptor at the kinetochore, the Rod-Zw10-Zwilch complex (RZZ). We found that RZZ and Spindly cooperate to form an outermost expanded kinetochore domain during cell division in human cultured cells, most likely by self-assembling into filamentous oligomers. Expansion of the kinetochore surface in early mitosis facilitates the capture of astral microtubules by dynein and other microtubule binding proteins present at the kinetochore, thereby accelerating the process of spindle assembly. Once kinetochores become attached to microtubules, dynein transports RZZ-Spindly and other outermost kinetochore components towards spindle poles as part of a motor-cargo complex, which contributes to the disassembly of the expanded kinetochore domain. This leaves a compact and microtubule-attached core kinetochore in place for the segregation of chromosomes to daughter cells. Our results, along with concurrent studies from other groups, define a new role for the RZZ-Spindly-dynein module as a key regulator of adaptive kinetochore size that enhances efficiency and accuracy of spindle assembly.
Second, we asked how the different subunits of the mega-dalton dynactin complex contribute to the function of this essential dynein co-factor. In the dividing C. elegans early embryo, we found that dynactin's microtubule binding activity, which resides in its largest subunit p150, helps dynein pull on microtubules to position the microtubule organizing center, the centrosome. We also discovered that the peripheral dynactin subunit p27 is required for targeting dynein-dynactin to kinetochores but not to other subcellular sites. This shows that while dynactin is a general activator of dynein motility, some of its subunits participate in specific cellular processes.
Third, we sought to identify new dynein pathway genes in a genome-wide RNAi screen in C. elegans. We used animals in which dynein function is partially compromised because the gene coding for one its co-factors, NudE, is lacking. We looked for genes whose downregulation of expression made NudE knockout animals more sick than control animals. This enhancer screen identified several dozen genes, many of which have functions in cell division. Characterization of two of the genes showed how their corresponding proteins co-operate with NudE at the kinetochore to ensure faithful chromosome segregation. How other genes identified in the screen relate to the dynein pathway will be interesting to investigate in the future.