Mitotic spindles are highly organized, microtubule (MT)-based, transient structures that serve the fundamental function of unerring chromosome segregation during cell division and thus of genomic stability during tissue morphogenesis and homeostasis. Hence, a multitude of MT-associated proteins (MAPs) regulates the dynamic assembly of MTs in preparation for mitosis. Some tumor suppressors, normally functioning to prevent tumor development, have now emerged as significant MAPs. Among those, neurofibromin, the product of the Neurofibromatosis-1 gene (NF1), a major Ras GTPase activating protein (RasGAP) in neural cells, controls also the critical function of chromosome congression in astrocytic cellular contexts. Cell type- and development-regulated splicings may lead to the inclusion or exclusion of NF1exon51, which bears a nuclear localization sequence (NLS) for nuclear import at G2; yet the functions of the produced NLS and ΔNLS neurofibromin isoforms have not been previously addressed. By using a lentiviral shRNA system, we have generated glioblastoma SF268 cell lines with conditional knockdown of NLS or ΔNLS transcripts. In dissecting the roles of NLS or ΔNLS neurofibromins, we found that NLS-neurofibromin knockdown led to increased density of cytosolic MTs but loss of MT intersections, anastral spindles featuring large hollows and abnormal chromosome positioning, and finally abnormal chromosome segregation and increased micronuclei frequency. Therefore, we propose that NLS neurofibromin isoforms exert prominent mitotic functions.
Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.