Tumor cells of a particular tissue may show a pattern of gene expression characteristic of the precursor cells of this tissue. To test this proposition for tumors of the central nervous system (CNS) we have used immunohistochemistry to analyze the expression of nestin in primary human CNS tumors and corresponding nonneoplastic brain tissue. Nestin defines a recently discovered sixth class of intermediate filament proteins and in the rat is expressed predominantly in CNS stem cells. In the adult nonneoplastic human brain we have detected only nestin expression in occasional endothelial cells. In contrast, a variety of primary CNS tumors contained substantially elevated nestin levels. The nestin-positive cells in the tumor tissue were tumor cells and/or endothelial cells. Glioblastomas expressed higher nestin levels than less malignant gliomas. This may indicate a correlation between nestin expression and malignancy within the glioma tumor group. In the primitive neuroectodermal class of tumors we observed both nestin-expressing and nonexpressing tumors, suggesting that nestin expression could be used to further characterize this complex and heterogeneous tumor group. Nine metastatic carcinomas were studied, and none showed nestin immunoreactivity in tumor cells. In conclusion, our data support the notion that primary CNS tumors share gene expression patterns with primitive, undifferentiated CNS cells and that nestin, like other intermediate filaments, may be useful in tumor diagnosis.

In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.

The assembly of intermediate filament (IF) arrays involves the recruitment of a complex set of cell-type-specific IF-associated proteins. Some of them are integral membrane proteins, others act as crosslinking proteins with vectorial binding activities, and yet others comprise motor proteins. In vivo IFs appear to be predominantly heteropolymers, although in vitro several IF proteins (e.g. vimentin, desmin, neurofilament (NF)-L and the nuclear lamins) do self-assemble into IF-like polymers. In contrast, NF-M, NF-H, nestin, synemin and paranemin, all bona fide IF proteins, are unable to self-assemble into IFs either in vitro or in vivo. The individual IF proteins of this large multigene family are chemically heterogeneous, exhibiting different assembly kinetics and yielding discrete types of filaments. The unique physical properties and interaction capabilities of these distinct IF molecular building blocks, in combination with accessory proteins, mediate the generation of a highly dynamic and interconnected, cell-type-specific cytoarchitecture.

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal-associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease.

BACKGROUND

This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24.

Disorganization of the neurofilament network is a prominent feature of several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy and axonal Charcot-Marie-Tooth disease. Giant axonal neuropathy (GAN, MIM 256850), a severe, autosomal recessive sensorimotor neuropathy affecting both the peripheral nerves and the central nervous system, is characterized by neurofilament accumulation, leading to segmental distension of the axons. GAN corresponds to a generalized disorganization of the cytoskeletal intermediate filaments (IFs), to which neurofilaments belong, as abnormal aggregation of multiple tissue-specific IFs has been reported: vimentin in endothelial cells, Schwann cells and cultured skin fibroblasts, and glial fibrillary acidic protein (GFAP) in astrocytes. Keratin IFs also seem to be alterated, as most patients present characteristic curly or kinky hairs. We report here identification of the gene GAN, which encodes a novel, ubiquitously expressed protein we have named gigaxonin. We found one frameshift, four nonsense and nine missense mutations in GAN of GAN patients. Gigaxonin is composed of an amino-terminal BTB (for Broad-Complex, Tramtrack and Bric a brac) domain followed by a six kelch repeats, which are predicted to adopt a beta-propeller shape. Distantly related proteins sharing a similar domain organization have various functions associated with the cytoskeleton, predicting that gigaxonin is a novel and distinct cytoskeletal protein that may represent a general pathological target for other neurodegenerative disorders with alterations in the neurofilament network.

The cytoskeletal lesions associated with enteropathogenic Escherichia coli adhering to cultured HeLa epithelial cells were examined by immunofluorescence microscopy. The microfilament-associated proteins actin, alpha-actinin, talin, and ezrin were localized with adherent enteropathogenic E. coli, whereas tropomyosin, keratin and vimentin (intermediate filaments), tubulin (microtubules), and vinculin were not localized. These cytoskeletal structures differed significantly from those associated with Salmonella typhimurium internalization (invasion).

A clone encoding mouse glial fibrillary acidic protein (GFAP) was isolated from a cDNA library constructed so as to express the cloned sequences. The library was screened using a GFAP-specific polyclonal antiserum; a single bacterial colony expressing GFAP was identified. The complete sequence of the cDNA insert in this clone is presented, encompassing 2.5 kilobases and specifying greater than 97% of the GFAP amino acid sequence. The clone includes a long (1.4-kilobase) 3' untranslated region. Within the coding region, the data show extensive homology with other intermediate filament proteins, particularly in those regions predicted to be alpha-helical. RNA blot transfer experiments using the cloned GFAP cDNA probe revealed a single GFAP mRNA species of 2.7 kilobases in mouse brain. Southern blot analysis indicates the existence of at most two genes encoding GFAP in the mouse genome. The mouse GFAP probe cross-hybridizes weakly at high stringency with genomic DNA from diverse eukaryotic species.

Mutations in the human Cu,Zn superoxide dismutase gene (SOD1) are found in 20% of kindreds with familial amyotrophic lateral sclerosis. Transgenic mice (line G1H) expressing a human SOD1 containing a mutation of Gly-93 ->> Ala (G93A) develop a motor neuron disease similar to familial amyotrophic lateral sclerosis, but transgenic mice (line N1029) expressing a wild-type human SOD1 transgene do not. Because neurofilament (NF)-rich inclusions in spinal motor neurons are characteristic of amyotrophic lateral sclerosis, we asked whether mutant G1H and/or N1029 mice develop similar NF lesions. NF inclusions (i.e., spheroids, Lewy body-like inclusions) were first detected in spinal cord motor neurons of the G1H mice at 82 days of age about the time these mice first showed clinical evidence of disease. Other neuronal intermediate filament proteins (alpha-internexin, peripherin) also accumulated in these spheroids. The onset of accumulations of ubiquitin immunoreactivity in the G1H mice paralleled the emergence of vacuoles and NF-rich spheroids in neurons, but they did not colocalize exclusively with spheroids. In contrast, NF inclusions were not seen in the N1029 mice until they were 132 days old, and ubiquitin immunoreactivity was not increased in the N1029 mice even at 199 days of age. Astrocytosis in spinal cord was associated with a marked increase in glial fibrillary acidic protein immunoreactivity in the G1H mice, but not in the N1029 mice. Finally, comparative studies revealed a striking similarity between the cytoskeletal pathology in the G1H transgenic mice and in patients with amyotrophic lateral sclerosis. These findings link a specific SOD1 mutation with alterations in the neuronal cytoskeleton of patients with amyotrophic lateral sclerosis. Thus, neuronal cytoskeletal abnormalities may be implicated in the pathogenesis of human familial amyotrophic lateral sclerosis.

The obligate intracellular bacterial pathogen Chlamydia trachomatis replicates within a large vacuole or "inclusion" that expands as bacteria multiply but is maintained as an intact organelle. Here, we report that the inclusion is encased in a scaffold of host cytoskeletal structures made up of a network of F-actin and intermediate filaments (IF) that act cooperatively to stabilize the pathogen-containing vacuole. Formation of F-actin at the inclusion was dependent on RhoA, and its disruption led to the disassembly of IFs, loss of inclusion integrity, and leakage of inclusion contents into the host cytoplasm. In addition, IF proteins were processed by the secreted chlamydial protease CPAF to form filamentous structures at the inclusion surface with altered structural properties. We propose that Chlamydia has co-opted the function of F-actin and IFs to stabilize the inclusion with a dynamic, structural scaffold while minimizing the exposure of inclusion contents to cytoplasmic innate immune-surveillance pathways.

Functional studies on the alpha6beta4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha6beta4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha6beta4 but no alpha6beta1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha6beta4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha6beta4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha6beta4 integrin and F-actin was seen. An association between alpha6beta4 and F-actin is supported by the fact that alpha6beta4 integrin and actin were released from clone A cells by treatment with the F-actin- severing protein gelsolin and that alpha6beta4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha6beta4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha6beta4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha6-specific antibody. Together, these results indicate that the alpha6beta4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.

Using both electron microscopy and immunological methods, we have characterized a number of changes occurring in rat fibroblasts after heat-shock treatment. Incubation of the cells for 3 h at 42 degrees-43 degrees C resulted in a number of changes within the cytoplasm including: a disruption and fragmentation of the Golgi complex; a modest swelling of the mitochondria and subtle alterations in the packing of the cristae; and alterations in cytoskeletal elements, specifically a collapse and aggregation of the vimentin-containing intermediate filaments around the nucleus. A number of striking changes were also found within the nuclei of the heat-treated cells: (a) We observed the appearance of rod-shaped bodies consisting of densely packed filaments. Using biochemical and immunological methods, these nuclear inclusion bodies were shown to be comprised of actin filaments. (b) Considerable alterations in the integrity of the nucleoli were observed after the heat-shock treatment. Specifically, there appeared to be a general relaxation in the condensation state of the nucleoli, changes in both the number and size of the granular ribonucleoprotein components, and finally a reorganization of the nucleolar fibrillar reticulum. These morphological changes in the integrity of the nucleoli are of significant interest since previous work as well as studies presented here show that two of the mammalian stress proteins, the major stress-induced 72-kD protein and the 110-kD protein, localize within the nucleoli of the cells after heat-shock treatment. We discuss these morphological changes with regards to the known biological and biochemical events that occur in cells after induction of the stress response.

We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N-cadherin which are thought to be important in cell-cell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF-L, alpha-internexin, NF-M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF-M. By 10-14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of nestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.

Using monoclonal antibodies, we demonstrate that the phosphoprotein of measles virus and a protein of herpes simplex virus type 1 crossreact with an intermediate filament protein of human cells. This intermediate filament protein, probably vimentin, has a molecular weight of 52,000, whereas the molecular weights of the measles viral phosphoprotein and the herpes virus protein are 70,000 and 146,000, respectively. Crossreactivity was shown by immunofluorescent staining of infected and uninfected cells and by immunoblotting. The monoclonal antibody against measles virus phosphoprotein did not react with herpes simplex virus protein and vice versa, indicating that these monoclonal antibodies recognize different antigenic determinants on the intermediate filament molecule. The significance of these results in explaining the appearance of autoantibodies during virus infections in humans is discussed.

Monoclonal antibodies generated against different human intermediate filament (IF) proteins were assayed on fixed, embedded tissue by the biotin-avidin-immunoperoxidase method for evaluation of the tissue specificity of these antibodies. An antibody (43 beta E8) made to fibroblast IF protein stains mesenchymal tissue such as endothelium, histiocytes, stromal fibroblasts, and Schwann cells but does not stain epithelium, skeletal muscle, lymphocytes, or neurons. Three different anti-cytokeratin antibodies decorate epithelium in three unique patterns. One (35 beta H11) stains all nonsquamous epithelium but fails to recognize squamous epithelium. Antibody 34 beta E12 stains the full thickness of squamous epithelium and ductular epithelium but does not react with hepatocytes, pancreatic acinar cells, proximal renal tubules, or endometrial glands. Antibody 34 beta B4 stains only the suprabasal portion of squamous epithelium. None of these three anti-cytokeratin antibodies reacts with nerve or mesenchymal tissue. Two anti-neurofilament antibodies recognize only neurons, failing to react with epithelial or mesenchymal tissue. We conclude that these anti-intermediate filament antibodies can be used as tissue-specific markers. Neoplasms retain the same intermediate filament patterns as the normal parental tissue; therefore, these antibodies can be used as diagnostic aids in surgical pathology.

Oncolytic herpes simplex virus-1 (HSV-1) mutants possessing mutations in the ICP34.5 and ICP6 genes have proven safe through clinical trials. However, ICP34.5-null viruses may grow poorly in cells due to their inability to prevent host-cell shut-off of protein synthesis caused by hyperphosphorylation of eukaryotic initiation factor 2alpha. To increase tumor selectivity, glioma-selective expression of ICP34.5 in the context of oncolysis may be useful. Malignant gliomas remain an incurable disease. One molecular marker of malignant gliomas is expression of the intermediate filament nestin. Expression of nestin mRNA was confirmed in 6 of 6 human glioma lines and in 3 of 4 primary glioma cells. Normal human astrocytes were negative. A novel glioma-selective HSV-1 mutant (rQNestin34.5) was thus engineered by expressing ICP34.5 under control of a synthetic nestin promoter. Replication, cellular propagation, and cytotoxicity of rQNestin34.5 were significantly enhanced in cultured and primary human glioma cell lines compared with control virus. However, replication, cellular propagation, and cytotoxicity of rQNestin34.5 in normal human astrocytes remained quantitatively similar to that of control virus. In glioma cell lines infected with rQNestin34.5, the level of phospho-eukaryotic initiation factor 2alpha was lower than that of cells infected by control rHsvQ1, confirming selective ICP34.5 expression in glioma cells. In vivo, rQNestin34.5 showed significantly more potent inhibition of tumor growth compared with control virus. Treatment in the brain tumor model was instituted on animal's display of neurologic symptoms, which usually led to rapid demise. rQNestin34.5 treatment doubled the life span of these animals. These results show that rQNestin34.5 could be a potent agent for the treatment of malignant glioma.

In recent years it has been shown that bacteria contain a number of cytoskeletal structures. The bacterial cytoplasmic elements include homologs of the three major types of eukaryotic cytoskeletal proteins (actin, tubulin, and intermediate filament proteins) and a fourth group, the MinD-ParA group, that appears to be unique to bacteria. The cytoskeletal structures play important roles in cell division, cell polarity, cell shape regulation, plasmid partition, and other functions. The proteins self-assemble into filamentous structures in vitro and form intracellular ordered structures in vivo. In addition, there are a number of filamentous bacterial elements that may turn out to be cytoskeletal in nature. This review attempts to summarize and integrate the in vivo and in vitro aspects of these systems and to evaluate the probable future directions of this active research field.

PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

The glutamate transporter GLAST is localized on the cell membrane of mature astrocytes and is also expressed in the ventricular zone of developing brains. To characterize and follow the GLAST-expressing cells during development, we examined the mouse spinal cord by in situ hybridization and immunohistochemistry. At embryonic day (E) 11 and E13, cells expressing GLAST mRNA were present only in the ventricular zone, where GLAST immunoreactivity was associated with most of the cell bodies of neuroepithelial cells. In addition, GLAST immunoreactivity was detected in radial processes running through the mantle and marginal zones. From this characteristic cytology, GLAST-expressing cells at early stages were judged to be radial glia cells. At E15, cells expressing GLAST mRNA first appeared in the mantle zone, and GLAST-immunopositive punctate or reticular protrusions were formed along the radial processes. From E18 to postnatal day (P) 7, GLAST mRNA or its immunoreactivity gradually decreased from the ventricular zone and disappeared from radial processes, whereas cells with GLAST mRNA spread all over the mantle zone and GLAST-immunopositive punctate/reticular protrusions predominated in the neuropils. At P7, GLAST-expressing cells were immunopositive for glial fibrillary acidic protein, an intermediate filament specific to astrocytes. Therefore, the glutamate transporter GLAST is expressed from radial glia through astrocytes during spinal cord development. Furthermore, the distinct changes in the cell position and morphology suggest that both the migration and transformation of radial glia cells begin in the spinal cord between E13 and E15, when the active stage of neuronal migration is over.

We have investigated the functional role of the non-helical end domains of vimentin on its assembly properties using truncated Xenopus and human recombinant proteins. Removal of the amino-terminal "head" domain yielded a molecule that did not assemble into 10 nm filaments but remained in a soluble oligomeric particle form with a sedimentation coefficient considerably smaller than that of wild-type vimentin (Vim(wt)). In contrast, removal of the carboxy-terminal "tail" domain had no obvious effect on the sedimentation characteristics. In particular, sedimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of Mr 135,000 to 360,000, indistinguishable from those obtained with Vim(wt). When induced to form filaments from this state by rapid dilution into filament forming buffer, Vim(wt) and Vim(deltaT) protein generated similar viscosity profiles. However, as determined by scanning transmission electron microscopy, under these conditions Vim(deltaT) formed filaments of heterogeneous diameter, corresponding to various distinct mass-per-length (MPL) values: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa/nm, Vim(deltaT) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contrast, when dialyzed against, instead of being rapidly diluted into, filament forming buffer, Vim(deltaT) gave histograms with one major peak at about 54 kDa/nm. The MPL heterogeneity observed for Vim(deltaT) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, "unit-length" filament segments (58 to 63 nm) were formed with both wt and deltaT proteins, but the diameters were considerably larger for Vim(deltaT) compared to Vim(wt) (20(+/- 3) nm versus 16(+/- 3)nm), indicating a distinct role of the carboxy-terminal tail domain in the width control during unit-length filament formation. Despite this difference both Vim(deltaT) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principally similar mechanism involving the end-on-fusion or annealing of unit-length filaments.