Nestin Intermediate Filament Research Articles

Engagement of vimentin intermediate filaments in hypotonic stress.

Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano-responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo-osmotic-induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo-osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin-1 (KIF5B) block the hypo-osmotic shock-induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin-dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo-osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.

Vimentin intermediate filaments function as a physical barrier during intracellular trafficking of caveolin-1

Both the cytoskeletal intermediate filaments (IFs) and cytoplasmic caveolae contribute to active processes such as cell migration, morphogenesis and vesicular trafficking, but the interplay between these two systems has remained elusive. Here, we find that vimentin and nestin IFs interact with caveolae central component caveolin-1 (CAV-1) and importantly, restrain the intracellular trafficking of CAV-1 positive vesicles by serving as a physical barrier. Consequently, CAV-1 vesicles show less density and mobility in vimentin IFs enriched region, which is a substrate stiffness independent process. Moreover, depletion of vimentin IFs releases the slow movement proportion of CAV-1 positive vesicles and thus increases their cytoplasmic dynamics, whereas the expression of caveolae-associated protein CAV-1, CAV-2 and cavin-1 were unaffected. Collectively, these results reveal a negative role of IFs in regulating the trafficking of intracellular CAV-1 vesicles in live cells.

Genome wide assessment of mRNA in astrocyte protrusions by direct RNA sequencing reveals mRNA localization for the intermediate filament protein nestin

Subcellular RNA localization plays an important role in development, cell differentiation, and cell migration. For a comprehensive description of the population of protrusion localized mRNAs in astrocytes we separated protrusions from cell bodies in a Boyden chamber and performed high-throughput direct RNA sequencing. The mRNAs with localization in astrocyte protrusions encode proteins belonging to a variety of functional groups indicating involvement of RNA localization for a palette of cellular functions. The mRNA encoding the intermediate filament protein Nestin was among the identified mRNAs. By RT-qPCR and RNA FISH analysis we confirmed Nestin mRNA localization in cell protrusions and also protrusion localization of Nestin protein. Nestin mRNA localization was dependent of Fragile X mental retardation syndrome proteins Fmrp and Fxr1, and the Nestin 3'-UTR was sufficient to mediate protrusion mRNA localization. The mRNAs for two other intermediate filament proteins in astrocytes, Gfap and Vimentin, have moderate and no protrusion localization, respectively, showing that individual intermediate filament components have different localization mechanisms. The correlated localization of Nestin mRNA with Nestin protein in cell protrusions indicates the presence of a regulatory mechanism at the mRNA localization level for the Nestin intermediate filament protein with potential importance for astrocyte functions during brain development and maintenance.

Protein Kinase Cζ Regulates Cdk5/p25 Signaling during Myogenesis

Atypical protein kinase Czeta (PKCzeta) is emerging as a mediator of differentiation. Here, we describe a novel role for PKCzeta in myogenic differentiation, demonstrating that PKCzeta activity is indispensable for differentiation of both C2C12 and mouse primary myoblasts. PKCzeta was found to be associated with and to regulate the Cdk5/p35 signaling complex, an essential factor for both neuronal and myogenic differentiation. Inhibition of PKCzeta activity prevented both myotube formation and simultaneous reorganization of the nestin intermediate filament cytoskeleton, which is known to be regulated by Cdk5 during myogenesis. p35, the Cdk5 activator, was shown to be a specific phosphorylation target of PKCzeta. PKCzeta-mediated phosphorylation of Ser-33 on p35 promoted calpain-mediated cleavage of p35 to its more active and stable fragment, p25. Strikingly, both calpain activation and the calpain-mediated cleavage of p35 were shown to be PKCzeta-dependent in differentiating myoblasts. Overall, our results identify PKCzeta as a controller of myogenic differentiation by its regulation of the phosphorylation-dependent and calpain-mediated p35 cleavage, which is crucial for the amplification of the Cdk5 activity that is required during differentiation.

Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.

Fhos2, a novel formin‐related actin‐organizing protein, probably associates with the nestin intermediate filament

Fhos1 is a mammalian formin-family protein, and functions as an organizer of the actin microfilament. Here we have cloned human and mouse cDNAs for a novel Fhos homolog, designated Fhos2. The messages for Fhos2 are expressed in the heart, kidney, and brain, where the Fhos1 mRNAs are not abundant. Two splice variants of Fhos2 exist in a tissue-specific manner; the longer variant Fhos2L is the major form in the heart, whereas the kidney and brain predominantly express Fhos2S that encodes a shorter protein. Over-expression of an active form of the two Fhos2 variants, as well as that of Fhos1, induces the formation of actin stress fibers in HeLa cells, suggesting that Fhos2 acts as an actin-organizing protein. Biochemical analysis using rat cardiomyoblastic H9c2 (2-1) cells reveals that endogenous Fhos2 is enriched in the intermediate filament fraction. Consistent with this, Fhos2 localizes to the nestin intermediate filament but not to other cytoskeletons, as demonstrated by staining of H9c2 (2-1) cells with anti-Fhos2 antibodies. Furthermore, Fhos2 is present in nestin-expressing neuroepithelial cells of the fetal rat brain. Thus, Fhos2 not only has the actin-organizing activity but also associates with nestin, which may imply a Fhos2-mediated link between the nestin intermediate filament and actin microfilament.

174 A phase II study of capecitabine in patients who have failed first-line treatment for locally advanced or metastatic cervix cancer (MCC)

The spatial and temporal immunoexpression of the intermediate filament (IF) protein nestin and its relationship to glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and its receptor flt-1 (VEGF-R1) in reactive astroglia was examined following stab wounds or transplants of fetal CNS tissue into the adult brain. Since developmentally regulated proteins and gene transcripts can be reexpressed in reactive astroglia following certain brain injuries, we analyzed the nestin profile in these experimental paradigms in order to more fully understand the nature of the gliotic “scar.” Nestin expression was transiently up-regulated in some but not all astrocytes which often had a different morphology than the typical stout, stellate GFAP (+) cells; the processes of the nestin (+) cells tended to be slender and elongated. In reactive astroglia from the mature brain, nestin expression was robust but generally localized to the wound or graft site, peaked at 7–10 days postoperative, and was absent by 28 days, whereas GFAP (+) astrocytes were far more widespread and persisted for many months. Only nestin was strongly expressed immediately adjacent to early stab wounds, whereas GFAP (+) cells were located further from the wound sites. In contrast, there was marked nestin/GFAP colocalization at the graft/host interface. Semiquantitative analysis combined with confocal microscopy revealed a unique compartmentalization of protein expression; processes from single astrocytes could be entirely nestin (+), GFAP (+), or could show coexpression. At 4, 7, and 14 days postoperative, 41, 58, and 32% of the immunoexpression, respectively, was accounted for by nestin at the graft/host interface, and it was essentially undetectable at 28 days postoperative. In situ hybridization studies showed nestin transcripts within GFAP (+) cells primarily between 4 and 10 days postoperative and absent by 28 days. Many nestin (+) astrocytes, as shown by electron microscopy, were closely related to the vasculature. Therefore we further examined the expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen associated with angiogenesis. Nestin colocalized with VEGF in some astrocytes (7%) but far more prominently with the VEGF flt-1 receptor (25%). Early astroglial activation may involve several different IF components and possibly a distinct astrocytic population that shows a rapid, transient nestin expression adjacent to injury sites. Expression of the nestin IF phenotype within affected astrocytes in the surgical vicinity may be indicative of a reversion to an immature phenotype that might be less susceptible to attendant hypoxia after injury. Since injured astrocytes are well known to express many bioactive compounds, such transient reexpression of early, developmentally regulated proteins may be a hallmark for the elaboration of growth factors such as VEGF.

Mouse nestin cDNA cloning and protein expression in the cytoskeleton of transfected cells

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

Transient coexpression of nestin, GFAP, and vascular endothelial growth factor in mature reactive astroglia following neural grafting or brain wounds.

The spatial and temporal immunoexpression of the intermediate filament (IF) protein nestin and its relationship to glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and its receptor flt-1 (VEGF-R1) in reactive astroglia was examined following stab wounds or transplants of fetal CNS tissue into the adult brain. Since developmentally regulated proteins and gene transcripts can be reexpressed in reactive astroglia following certain brain injuries, we analyzed the nestin profile in these experimental paradigms in order to more fully understand the nature of the gliotic "scar." Nestin expression was transiently up-regulated in some but not all astrocytes which often had a different morphology than the typical stout, stellate GFAP (+) cells; the processes of the nestin (+) cells tended to be slender and elongated. In reactive astroglia from the mature brain, nestin expression was robust but generally localized to the wound or graft site, peaked at 7-10 days postoperative, and was absent by 28 days, whereas GFAP (+) astrocytes were far more widespread and persisted for many months. Only nestin was strongly expressed immediately adjacent to early stab wounds, whereas GFAP (+) cells were located further from the wound sites. In contrast, there was marked nestin/GFAP colocalization at the graft/host interface. Semiquantitative analysis combined with confocal microscopy revealed a unique compartmentalization of protein expression; processes from single astrocytes could be entirely nestin (+), GFAP (+), or could show coexpression. At 4, 7, and 14 days postoperative, 41, 58, and 32% of the immunoexpression, respectively, was accounted for by nestin at the graft/host interface, and it was essentially undetectable at 28 days postoperative. In situ hybridization studies showed nestin transcripts within GFAP (+) cells primarily between 4 and 10 days postoperative and absent by 28 days. Many nestin (+) astrocytes, as shown by electron microscopy, were closely related to the vasculature. Therefore we further examined the expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen associated with angiogenesis. Nestin colocalized with VEGF in some astrocytes (7%) but far more prominently with the VEGF flt-1 receptor (25%). Early astroglial activation may involve several different IF components and possibly a distinct astrocytic population that shows a rapid, transient nestin expression adjacent to injury sites. Expression of the nestin IF phenotype within affected astrocytes in the surgical vicinity may be indicative of a reversion to an immature phenotype that might be less susceptible to attendant hypoxia after injury. Since injured astrocytes are well known to express many bioactive compounds, such transient reexpression of early, developmentally regulated proteins may be a hallmark for the elaboration of growth factors such as VEGF.

Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class.

Our laboratory previously reported that transitin is a radial glial intermediate filament protein sharing the basic structural features common to all intermediate filament (IF) proteins. It contains an alpha-helical core domain flanked by a short nonhelical head and a long COOH-terminal tail. The core sequence of transitin shows the greatest similarity to Xenopus tanabin and to rat and human nestin. We also reported that transitin has multiple splice variants derived from the deletion or inclusion of a leucine-zipper heptad repeat domain in the COOH-terminal tail. In the present study, we provide new evidence to support the classification of nestin and transitin in the same group of IF proteins based on the number and position of its introns. In addition, we suggest that the different isoforms of transitin are produced by a splicing mechanism that recognizes consensus 5' and 3' splice sites contained within the coding sequence of the leucine-zipper heptad repeat domain.

Early patterns of migration, morphogenesis, and intermediate filament expression of subventricular zone cells in the postnatal rat forebrain

Recent studies using retroviral labeling of subventricular zone (SVZ) progenitors in vivo in neonatal rats have directly demonstrated the generation of both astrocytes and oligodendrocytes from these progenitors. In the present study, we used a recombinant retroviral vector encoding beta-galactosidase, and analyzed brains within the first week after retroviral injection to trace the early routes that SVZ cells take as they migrate into white matter and cortex and characterized the early morphological and antigenic changes that accompanied their differentiation. SVZ cells follow specifically definable migratory routes as they colonize the cortex and subcortical white matter. Glial progenitors do not populate the cortex in a systematic, laminar fashion, as do neuroblasts. The abundance of labeled progenitors in radial arrangements and the close apposition of many immature cells to vimentin+ radial glial processes, suggest that glial progenitors migrate along radial glia. Labeled SVZ cells, which displayed a simple, unipolar or bipolar morphology, lacked detectable vimentin and nestin intermediate filaments. Similarly, beta-galactosidase-positive cells in white matter lacked these filaments. In contrast, labeled, multipolar cells in the cortex, and a few of the immature-appearing cortical cells expressed nestin and vimentin. At these early time points, GFAP was not detected in beta-galactosidase-labeled cells. Multipolar cells in cortex frequently displayed processes extending toward and contacting blood vessels. These observations suggest that the expression of nestin and vimentin occurs after progenitors emigrate from the SVZ and that filament expression and contact with blood vessels represent an early stage of astrocyte differentiation.

Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain

Proliferating precursors to the distinct cell types constituting the mammalian brain can be identified by the presence of the nestin intermediate filament. We report the establishment of a nestin-positive cell line, HiB5, from embryonic precursor cells to the rat hippocampus. Since it was immortalized using the temperature-sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 33°C, but not at 39°C, the body temperature of rodents. To test their developmental capacity, HiB5 cells were implanted into both the neonatal hippocampus and cerebellum. The cells integrated into the host tissue and acquired morphologies characteristic of the neurons and glial cells found at the implant site. HiB5 cells might thus be useful in characterizing the signals regulating cell type determination in the mammalian brain.