Nuclear Isoforms of Neurofibromin Are Required for Proper Spindle Organization and Chromosome Segregation

Abstract

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.

Two Tails for Neurofibromin: A Tale of Two Microtubule-Associated Proteins

“…In an earlier study with primary neurons, we found that neurofibromin, in addition to its colocalization with cytosolic MTs, localizes also in the nucleus and identified a bipartite NLS in the CTD (Figure 1B) as the probable mediator of nuclear entry [37]. Previously thought as an artifact in the skin epithelium, nuclear neurofibromin is detected with a variety of techniques, i.e., immunocytochemistry (e.g., white arrows in Figure 3), subcellular fractionations, or proteomics [28,29,[37][38][39][51][52][53] in all cells of an ectodermal origin.…”

“…The importance of NLS inclusion was totally unexplored, till we documented a few years ago that neurofibromin controls the pivotal function of chromosome congression on the mitotic spindle [38] (Figure 2) and then proved that depletion of NLS neurofibromins deregulates spindle assembly and positioning, leading to aneuploidy and increased micronuclei formation [39]. More importantly, these studies established the function of neurofibromins as MAPs.…”

“…Thus, we have postulated that neurofibromin primarily co-localizes with β-tubulin at all stages of spindle assembly from prophase to metaphase (e.g., Figure 2B) and then through the transformation of the spindle to a machinery for chromosome segregation and cell division, that's is through anaphase, telophase and cytokinesis [38,39]. Again, neurofibromin's localization onto the spindle is apparent in all cells of ectodermal origin, with no exception.…”

“…Association of neurofibromin with cytosolic MTs was first established by confocal microscopy in fibroblasts and the molecule was proposed to act as a MAP, through a small segment (residues 815-834 in the CSRD) bearing in silico homology to MAPs Tau and MAP2 [49]. Since then, diverse experimental approaches, including co-immunoprecipitations, co-purifications, in vitro MT assembly, and affinity precipitations, have further documented this interaction with cytosolic [23,24,29,37,38,50] and with mitotic MTs [38,39].…”

“…Indeed, confocal image analysis of primary or tumor cells derived from the ectoderm or the neural crest and quadruply stained for β-tubulin, neurofibromin, F-actin and chromatin/chromosomes, shows that pools of endogenous neurofibromin colocalize with cytoplasmic MTs (e.g., rat astrocytes in Figure 3A, yellow arrows), as well as with F-actin, mainly at the cell cortex and lamellipodia ( [37][38][39]; Figure 3B, yellow arrows). Interestingly, no association could be established with any intermediate filament in neural cells, as for example with the abundant astrocytic marker GFAP (Figure 3C), except for nuclear lamins [38].…”