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.
Highlights
The ability of tubulins to rapidly form highly dynamic noncovalent polymers, the microtubules (MTs), has bestowed on them essential functions for the constant yet ever changing needs for spatial organization of cells, in order to execute critical processes, such as attaining function-coupled shapes, directed intracellular transport, cell migration, and the most important for development and maintenance of an organism cell divisions with accurate genomic transmission
In order to observe the effects of nuclear localization sequence (NLS)- and ∆NLS- neurofibromin isoforms after knockdown of their respective Neurofibromatosis-1 gene (NF1) transcript (Scheme 1) over a long time and many cell divisions while increasing reproducibility of results, we chose to interfere with the mRNA production of NLS and ∆NLS NF1 transcripts [52], by generating for each transcript shRNA constructs, which are capable of DNA
These results strongly suggest that neurofibromin isoforms, changed only by the inclusion, or not, of an NLS sequence have opposing, possibly complementary when both expressed, functions, that are required for mitotic aster formation and spindle assembly for efficient chromosome congression and error correction, at least in an astrocytic cellular background
Summary
The ability of tubulins to rapidly form highly dynamic noncovalent polymers, the microtubules (MTs), has bestowed on them essential functions for the constant yet ever changing needs for spatial organization of cells, in order to execute critical processes, such as attaining function-coupled shapes, directed intracellular transport, cell migration, and the most important for development and maintenance of an organism cell divisions with accurate genomic transmission For the latter, several types of MTs organize, elongate, and orient a bipolar spindle, through which chromosomes will position at the spindle equator for faithful sister chromatid separation and segregation to the two daughter cells [1,2,3]. As aberrations in any of these processes may lead to aneuploidy and further on to tumorigenesis, the ability of MAPs to modulate MT dynamics is recognized for its prognostic value in cancers, and as a sensible target for manipulations of microtubule-targeting cancer chemotherapy agents [12,13]
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