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.

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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.

Understanding the long term impact of early life seizures (ELS) is of vital importance both for researchers and clinicians. Most experimental studies of how seizures affect the developing brain have drawn their conclusions based on changes detected at the cellular or behavioral level, rather than on intermediate levels of analysis, such as the physiology of neuronal networks. Neurons work as part of networks and network dynamics integrate the function of molecules, cells and synapses in the emergent properties of brain circuits that reflect the balance of excitation and inhibition in the brain. Therefore, studying network dynamics could help bridge the cell-to-behavior gap in our understanding of the neurobiological effects of seizures. To this end we investigated the long-term effects of ELS on local network dynamics in mouse neocortex. By using the pentylenetetrazole (PTZ)-induced animal model of generalized seizures, single or multiple seizures were induced at two different developmental stages (P9–15 or P19–23) in order to examine how seizure severity and brain maturational status interact to affect the brain’s vulnerability to ELS. Cortical physiology was assessed by comparing spontaneous network activity (in the form of recurring Up states) in brain slices of adult (>5 mo) mice. In these experiments we examined two distinct cortical regions, the primary motor (M1) and somatosensory (S1) cortex in order to investigate regional differences in vulnerability to ELS. We find that the effects of ELSs vary depending on (i) the severity of the seizures (e.g., single intermittent ELS at P19–23 had no effect on Up state activity, but multiple seizures induced during the same period caused a significant change in the spectral content of spontaneous Up states), (ii) the cortical area examined, and (iii) the developmental stage at which the seizures are administered. These results reveal that even moderate experiences of ELS can have long lasting age- and region-specific effects in local cortical network dynamics.

Neurofibromatosis type 1, NF-1, is a common monogenic (NF1) disease, characterized by highly variable clinical presentation and high predisposition for tumors, especially those of astrocytic origin (low- to high-grade gliomas). Unfortunately, very few genotype–phenotype correlations have been possible, and the numerous identified mutations do not offer help for prognosis and patient counselling. Whole gene deletion in animals does not successfully model the disease, as NF-1 cases caused by point mutations could be differentially affected by cell type-specific alternative splice variants of NF1. In this chapter, we will discuss the differential Microtubule-Associated-Protein (MAP) properties of NLS or ΔNLS neurofibromins, produced by the alternatively splicing of exon 51, which also contains a Nuclear Localization Sequence (NLS), in the assembly of the mitotic spindle and in faithful genome transmission. We will also commend on the major theme that emerges about NLS-containing tumor suppressors that function as mitotic MAPs.