Molecular And Proteomics Dissection Of Nuclear PML Body And PRC1 Central Spindle Organization

The nucleus was one of the first intracellular structures to be identified by microscopy, but its functional organization is still poorly understood. Interphase mammalian nuclei are compartmentalized into a large number of structures or organelles that are likely to contribute to the genomic stability and dynamics. Dramatic transformations in the structural organization of both nucleus and the cytoplasm occur during mitosis. The onset of mitosis is accompanied by chromatin condensation, breakdown of the nuclear envelope followed by chromosome segregation. During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. I will summarize my pursuits in understanding of nuclear dynamics in interphase and mitotic cells.PML nuclear bodies were first identified as nuclear domain 10 (ND10) by autoantibodies recognizing the Sp100 protein. The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells, resulting from a reciprocal chromosome translocation t (15; 17). My studies show that the nuclear localization of PML is regulated by SUMO-3, one of the three recently identified SUMO isoforms in human cells. SUMO-3 bears similar subcellular distribution to those of SUMO-1 and -2 in the interphase nuclear body, which is colocalized with PML protein. However, both SUMO-2 and -3 are also localized to nucleoli, a region lacking SUMO-1. Immunoprecipitated PML protein bears SUMO-3 moiety in a covalently modified form, supporting the notion that PML is conjugated by SUMO-3. To determine the functional relevance of SUMO-3 conjugation on PML molecular dynamics, we suppressed SUMO-3 protein expression using a siRNA-mediated approach. Depletion of SUMO-3 markedly reduced the number of PML-containing NBa and their integrity, which is rescued by exogenous expression of SUMO-3 but not SUMO-1 or SUMO-2. The specific requirement of SUMO-3 for PML nuclear localization is validated by expression of SUMO-3 conjugation defective mutant. Moreover, we demonstrate that oligomerization of SUMO-3 is required for PML retention in the nucleus. Taken together, our...