Vimentin Phosphorylation Is Required for Normal Cell Division of Immature Astrocytes.

Vimentin (VIM) is an intermediate filament (nanofilament) protein expressed in multiple cell types, including astrocytes. Mice with VIM mutations of serine sites phosphorylated during mitosis (VIMSA/SA) show cytokinetic failure in fibroblasts and lens epithelial cells, chromosomal instability, facilitated cell senescence, and increased neuronal differentiation of neural progenitor cells. Here we report that in vitro immature VIMSA/SA astrocytes exhibit cytokinetic failure and contain vimentin accumulations that co-localize with mitochondria. This phenotype is transient and disappears with VIMSA/SA astrocyte maturation and expression of glial fibrillary acidic protein (GFAP); it is also alleviated by the inhibition of cell proliferation. To test the hypothesis that GFAP compensates for the effect of VIMSA/SA in astrocytes, we crossed the VIMSA/SA and GFAP-/- mice. Surprisingly, the fraction of VIMSA/SA immature astrocytes with abundant vimentin accumulations was reduced when on GFAP-/- background. This indicates that the disappearance of vimentin accumulations and cytokinetic failure in mature astrocyte cultures are independent of GFAP expression. Both VIMSA/SA and VIMSA/SAGFAP-/- astrocytes showed normal mitochondrial membrane potential and vulnerability to H2O2, oxygen/glucose deprivation, and chemical ischemia. Thus, mutation of mitotic phosphorylation sites in vimentin triggers formation of vimentin accumulations and cytokinetic failure in immature astrocytes without altering their vulnerability to oxidative stress.

Nestin Regulates Neurogenesis in Mice Through Notch Signaling From Astrocytes to Neural Stem Cells.

The intermediate filament (nanofilament) protein nestin is a marker of neural stem cells, but its role in neurogenesis, including adult neurogenesis, remains unclear. Here, we investigated the role of nestin in neurogenesis in adult nestin-deficient (Nes-/-) mice. We found that the proliferation of Nes-/- neural stem cells was not altered, but neurogenesis in the hippocampal dentate gyrus of Nes-/- mice was increased. Surprisingly, the proneurogenic effect of nestin deficiency was mediated by its function in the astrocyte niche. Through its role in Notch signaling from astrocytes to neural stem cells, nestin negatively regulates neuronal differentiation and survival; however, its expression in neural stem cells is not required for normal neurogenesis. In behavioral studies, nestin deficiency in mice did not affect associative learning but was associated with impaired long-term memory.

Nuclear phosphatidylinositol 4,5-bisphosphate islets contribute to efficient RNA polymerase II-dependent transcription.

This paper describes a novel type of nuclear structure - nuclear lipid islets (NLIs). They are of 40-100 nm with a lipidic interior, and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] molecules comprise a significant part of their surface. Most of NLIs have RNA at the periphery. Consistent with that, RNA is required for their integrity. The NLI periphery is associated with Pol II transcription machinery, including the largest Pol II subunit, transcription factors and NM1 (also known as NMI). The PtdIns(4,5)P2-NM1 interaction is important for Pol II transcription, since NM1 knockdown reduces the Pol II transcription level, and the overexpression of wild-type NM1 [but not NM1 mutated in the PtdIns(4,5)P2-binding site] rescues the transcription. Importantly, Pol II transcription is dependent on NLI integrity, because an enzymatic reduction of the PtdIns(4,5)P2 level results in a decrease of the Pol II transcription level. Furthermore, about half of nascent transcripts localise to NLIs, and transcriptionally active transgene loci preferentially colocalise with NLIs. We hypothesize that NLIs serve as a structural platform that facilitates the formation of Pol II transcription factories, thus participating in the formation of nuclear architecture competent for transcription.

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice.

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.

Paxillin-dependent regulation of IGF2 and H19 gene cluster expression.

Paxillin (PXN) is a focal adhesion protein that has been implicated in signal transduction from the extracellular matrix. Recently, it has been shown to shuttle between the cytoplasm and the nucleus. When inside the nucleus, paxillin promotes cell proliferation. Here, we introduce paxillin as a transcriptional regulator of IGF2 and H19 genes. It does not affect the allelic expression of the two genes; rather, it regulates long-range chromosomal interactions between the IGF2 or H19 promoter and a shared distal enhancer on an active allele. Specifically, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, thus activating IGF2 gene transcription, whereas it restrains the interaction between the enhancer and the H19 promoter, downregulating the H19 gene. We found that paxillin interacts with cohesin and the mediator complex, which have been shown to mediate long-range chromosomal looping. We propose that these interactions occur at the IGF2 and H19 gene cluster and are involved in the formation of loops between the IGF2 and H19 promoters and the enhancer, and thus the expression of the corresponding genes. These observations contribute to a mechanistic explanation of the role of paxillin in proliferation and fetal development.

UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity.

To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1ß-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis.