NuMA assembles into an extensive filamentous structure when expressed in the cell cytoplasm

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects on the progression of mitosis. Examination of the cytoplasmic mass formed by NuMA-DeltaNLS using transmission electron microscopy (TEM) revealed an extensive network of approximately 5 nm filaments that are further organized by the presence of dynamic microtubules into a dense web of solid, approximately 23 nm cables. Using flow cytometry, we have isolated the intact filamentous mass formed by NuMA-DeltaNLS from lysates of transiently transfected cells. These isolated structures are constructed of networks of interconnected 5 nm filaments and are composed exclusively of NuMA. These data demonstrate that NuMA is capable of assembling into an extensive filamentous structure supporting the possibility that NuMA serves a structural function either in the nucleus during interphase or at the polar ends of the mitotic spindle.