III Intermediate Filament Research Articles

Targeting Vimentin (VIM) in Acute Myelogenous Leukemia (AML)

Background: Vimentin (VIM) is a type III intermediate filament (IF) that anchors organelles in the cell, maintains cell integrity, and facilitates mitosis. A 2018 study demonstrated that the up-regulation of VIM is associated with poor clinical outcome in patients with Acute Myeloid Leukemia (AML). This study also reported that VIM regulates signaling networks that promote leukemic cell survival while conferring resistance to various stressors. A separate study revealed that VIM over-expression was associated both with markedly shorter overall survival (OS) and leukemia-free survival (LFS) in adult AML patients. VIM has been shown to be targetable by the steroidal lactone Withaferin-A which directly binds VIM, hyper-phosphorylates VIM's Serine 38 residue, and leads to an anti-proliferative effect characterized by perturbation of intermediary filaments. We investigated VIM expression and prognostic impact in pediatric AML, and whether inhibiting VIM affected chemosensitivity in vitro. Methods: Reverse Phase Protein Array (RPPA) was performed to quantify relative expression of 233 total and 63 post-translational modified proteins in 500 pediatric AML patients from the COG AAML1031 trial. AML cell lines with low VIM expression (OCI-2) and high VIM expression (OCI-3) as well as fresh, primary AML samples were exposed to the VIM inhibitor Withaferin-A alone, or in combination with Doxorubicin and Cytarabine chemotherapy. Other known VIM inhibitors including FiVe1, Simvastatin, and Ganoderic Acid A were tested for efficacy in our context, but only Withaferin-A produced a promising anti-leukemia effect. Results: In pediatric AML, VIM was over-expressed relative to normal CD34+ cells in all subtypes: bulk, CD3/CD19 depleted, CD34+, CD34+/38+, CD34+/38-, CD34-, CD34-/38+, and CD34-/38-. Pearson correlation analysis revealed significant negative correlations between VIM and proteins known to decrease during metastasis such as PDGFR-beta, TP15.pS15, HIF1-alpha and VHL. Elevated VIM correlated positively with HNRNPK, WTAP, ASH2L, and KDM1A, and H3K4Me2; alterations in histone methylation that are associated with metastatic potency. VIM expression was highest in FAB subtypes M4 and M5, and in patients with t(9;11) and MLL-rearrangements. VIM levels were lower in cytogenetically favorable and higher in unfavorable risk groups. Cumulative survival probability to standard chemotherapy progressively decreased as VIM expression increased (Fig. 1). We tested whether VIM inhibition with Withaferin-A at 50 nM and 100 nM, would affect proliferation or cell survival, finding a significantly stronger anti-proliferative effect (p<0.02) in the high-VIM OCI-3 cell line compared to the low-VIM OCI-2 cell line at 72 and 96 hours after treatment (Fig. 2). Combined with chemotherapy, Withaferin-A lead to greater cell death in the high-VIM OCI-3 cell line as opposed to low-VIM OCI-2 at 72 and 96 hours. For instance, in OCI-3, 100 nM Withaferin-A combined separately with either 1 uM Cytarabine or 50 nM Doxorubicin to decrease proliferation more than either chemotherapeutic agent alone (p=0.007 and p=0.0013). In primary AML samples, proliferation was slightly more inhibited at 72 hours post-treatment when Withaferin-A was paired with individual chemotherapeutics. Conclusions: Proteomic analysis demonstrated that VIM was elevated in numerous leukemia cell subtypes relative to normal CD34+ cells in children and adults and that prognosis worsened as VIM levels increased in pediatric AML. AML cell lines highly expressing VIM such as OCI-3 displayed sensitivity to both Withaferin-A monotherapy and Withaferin-A in combination with Cytarabine and Doxorubicin. Finally, Withaferin-A significantly reduced viability and proliferation after 72 hours of treatment in primary AML samples at select doses in combination with chemotherapeutics. These results suggest that targeting VIM could improve the efficacy of chemotherapy in high VIM expressing subpopulations and potentially improve overall survival. Withaferin-A's efficacy both as a mono-therapeutic and in combination with standard chemotherapeutics used to treat AML patients offers an exciting new area of research that requires further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

New GFAP splice isoform (GFAPµ) differentially expressed in glioma translates into 21 kDa N-terminal GFAP protein.

The glial fibrillary acidic protein (GFAP) is a type III intermediate filament (IF) protein that is highly expressed in astrocytes, neural stem cells, and in gliomas. Gliomas are a heterogeneous group of primary brain tumors that arise from glia cells or neural stem cells and rely on accurate diagnosis for prognosis and treatment strategies. GFAP is differentially expressed between glioma subtypes and, therefore, often used as a diagnostic marker. However, GFAP is highly regulated by the process of alternative splicing; many different isoforms have been identified. Differential expression of GFAP isoforms between glioma subtypes suggests that GFAP isoform-specific analyses could benefit diagnostics. In this study we report on the differential expression of a new GFAP isoform between glioma subtypes, GFAPµ. A short GFAP transcript resulting from GFAP exon 2 skipping was detected by RNA sequencing of human glioma. We show that GFAPµ mRNA is expressed in healthy brain tissue, glioma cell lines, and primary glioma cells and that it translates into a ~21kDa GFAP protein. 21kDa GFAP protein was detected in the IF protein fraction isolated from human spinal cord as well. We further show that induced GFAPµ expression disrupts the GFAP IF network. The characterization of this new GFAP isoform adds on to the numerous previously identified GFAP splice isoforms. It emphasizes the importance of studying the contribution of IF splice variants to specialized functions of the IF network and to glioma research.

Transcriptional and Phenotypic Characterization of Vimentin Knockout Mouse during Aging

Vimentin is a type III intermediate filament (IF) that primarily found in mesenchymal cells and plays important roles in epithelial‐mesenchymal transition (EMT). IFs were originally thought to be a relatively static structure that mainly functioned as mechanical support. However, in the past decades, it has been shown that IF networks are highly dynamic structures and their roles have expanded from maintenance of cell shape and integrity to cellular migration, adhesion, and cancer metastasis. Vimentin knockout mouse was originally reported to have no overt phenotype, leading to the conclusion that vimentin IF represented an unnecessary cytoskeletal system. Interestingly, our group recently found that acute lung injury (ALI) is attenuated in mice lacking vimentin when challenged with either LPS, asbestos, or bleomycin. Moreover, these studies demonstrated vimentin to be a key player in the activation of the NACHT, LRR and PYD domains‐containing protein 3 (NLRP3) inflammasome, an intracellular protein complex that is activated in response to stimuli such as bacterial and viral pathogens, including influenza A virus. In this study, we thoroughly characterize young and aged vimentin knockout mice behaviorally using battery of neurocognitive tests and show that there are age‐dependent changes in neuromuscular, neurocognitive, and metabolic lung functions distinctively from the wild type mice. We also use RNA sequencing as an unbiased approach to study transcriptional changes and identify a significant number of differentially expressed genes between the two groups in various cell types and with aging. Additionally, histological analyses from multiple organs of these mice also suggest a crucial role of vimentin in maintaining cellular structures and functions during aging. In conclusion, our data suggests that vimentin intermediate filament plays dynamic roles in multiple organ systems and during aging.Support or Funding InformationNHLBI R01 HL128194This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Desmin deficiency is not sufficient to prevent corneal fibrosis

The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model.

Crystal structure of the human glial fibrillary acidic protein 1B domain

Glial fibrillary acidic protein (GFAP) is a homopolymeric type III intermediate filament (IF) that plays essential roles in cell migration, mitosis, development, and signaling in astrocytes and a specific type of glial cells. Its overexpression and genetic mutations lead to abnormal IF networks and accumulation of Rosenthal fibers, which results in the fatal neurodegenerative disorder Alexander disease. Herein, we present the first crystal structure of human GFAP spanning the central coiled-coil 1B domain at 2.5 Å resolution. The domain forms a tetramer comprising two equivalent parallel coiled-coil dimers that pack together in an antiparallel manner. Its assembly is stabilized by extensive networks of intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions. Furthermore, mapping of the GFAP mutations associated with Alexander disease reveals that most involve residues buried in the core of the interface, and are likely to disrupt the intermolecular interactions and/or introduce steric clashes, thereby decreasing GFAP solubility and promoting aggregation. Based on our structural analysis and previous biochemical studies, we propose that GFAP assembles in the A11 mode in which coiled-coil 1B dimers lie in close axial proximity in an antiparallel fashion to provide a stable tetrameric platform for the organization of the GFAP filament.

Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin.

SummaryType III intermediate filament (IF) proteins assemble into cytoplasmic homopolymeric and heteropolymeric filaments with other type III and some type IV IFs. These highly dynamic structures form an integral component of the cytoskeleton of muscle, brain, and mesenchymal cells. Here, we review the current ideas on the role of type III IFs in health and disease. It turns out that they not only offer resilience to mechanical strains, but, most importantly, they facilitate very efficiently the integration of cell structure and function, thus providing the necessary scaffolds for optimal cellular responses upon biochemical stresses and protecting against cell death, disease, and aging.

Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues

Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo.

Imaging Withaferin A‐Vimentin Interactions in Live Cells and Fibrotic Tissues

The objective of this study was to illuminate the targeting of vimentin by the anti‐fibrotic natural product withaferin A (WFA). Here we have developed a novel WFA‐BODIPY‐FL imaging reagent (WFA‐Verde) to probe the functions of type III intermediate filament (IF) protein vimentin, a prominent biomarker of fibrotic cells and invasive cancer cells. WFA‐Verde exhibits high cell permeability demonstrating rapid entry into cultures of baby hamster kidney‐1 (BHK‐1) cells and rabbit corneal fibroblasts (RCFs). The staining pattern of WFA‐Verde in RCFs cells treated for 5 min and then fixed showed diffuse cytoplasmic fluorescence, which over time showed temporal incorporation of the probe into the filamentous vimentin forms by 30–60 min post treatment. Since WFA targets soluble vimentin and causes increased phosphorylation at serine 38 residue, we investigated also the staining characteristics of RCFs treated with drug‐like higher concentrations of WFA‐Verde. Subconfluent RCFs treated for 1 h, washed and left in culture medium showed recovery of cell shape and cytoskeleton by 18 hours. Cells stained for phosphorylated serine 38 vimentin showed extensive overlap with BODIPY‐labeled fluorescence that clearly illuminated individual filaments throughout the cell cytoplasm. Next, to investigate early kinetics of polymerization of soluble vimentin into filamentous cytoskeleton we employed a cell spreading assay. BHK‐1 cells were trypsinized, plated for 1 h and treated with 250 nM WFA‐Verde for 5 min, washed and immediately imaged every 5 seconds for 10–12 mins. In these live‐cell imaging experiments we found the bulk of soluble vimentin labeled was perinuclear and newly formed filaments precursors (represented by dots, squiggles and looping structures) showed dynamic movements similar to that reported with hybrid cDNA constructs containing green fluorescent protein‐vimentin in transfection studies. Dots‐like structures moved bilaterally and some dots moved at speeds of ~0.5 micrometers/sec, which is in the range previously reported. Since WFA exhibits potent ant‐fibrotic activity by targeting type III IFs, we next investigated whether WFA‐Verde is also incorporated into fibrotic tissues in vivo. Employing a model of ocular alkali‐injury that results in corneal fibrosis due to abundant overexpression of vimentin in myofibroblasts, we investigated the in vivo delivery of WFA‐Verde on corneal staining in eyes of uninjured mice and from those injured for 14 days. The fluorescent reagent within 1 hour of delivery achieved tissue permeabilization and accumulated abundantly in corneal myofibroblasts that overexpress vimentin as corroborated by staining overlap with anti‐vimentin antibody as well as anti‐phosphorylated serine 38 vimentin antibody. The uninjured eye sections due to very low expression of vimentin did not show comparable strong WFA‐Verde fluorescence as that observed with injured eyes. Dose response studies and time course investigations are currently being conducted to optimize target labeling. Thus, in conclusion we have developed the first‐in‐class imaging probe for vimentin that binds and labels vimentin in cells and in vivo. The use of WFA‐Verde as an imaging reagent for fibrosis biomarker detection in live animals is being currently pursued as this will afford us a non‐invasive means to image disease and injury ultimately in humans.Support or Funding InformationThis work was funded by NIH grant R01EY16782 and by the John A. and Florence Mattern Solomon Endowed Chair in Vision Biology and Eye Diseases.

The Use of Withaferin A to Study Intermediate Filaments.

Withaferin A (WFA), initially identified as a compound that inhibits experimental angiogenesis, has been shown to bind to soluble vimentin (sVim) and other type III intermediate filament (IF) proteins. We review WFA's dose-related activities (Section 1), examining nanomolar concentrations effects on sVim in cell proliferation and submicromolar effects on lamellipodia and focal adhesion formation. WFA effects on polymeric IFs are especially interesting to the study of cell migration and invasion that depend on IF mechanical contractile properties. WFA interferes with NF-κB signaling, though this anti-inflammatory mechanism may occur via perturbation of sVim-protein complexes, and possibly also via targeting IκB kinase β directly. However, micromolar concentrations that induce vimentin cleavage to promote apoptosis may increasingly show off-target effects via targeting other IFs (neurofilaments and keratin) and non-IFs (tubulin, heat-shock proteins, proteasome). Thus, in Section 2, we describe our studies combining cell cultures with animal models of injury to validate relevant type III IF-targeting mechanisms of WFA. In Section 3, we illuminate from investigating myofibroblast differentiation how sVim phosphorylation may govern cell type-selective sensitivity to WFA, offering impetus for exploring vimentin phosphorylation isoforms as targets and biomarkers of fibrosis. These different WFA targets and activities are listed in a summary table.

Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM(SA/SA)) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM(WT/SA)) were indistinguishable from WT (VIM(WT/WT)) mice. In VIM(SA/SA) mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM(SA/SA), reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM(SA/SA), the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.

Withaferin A effectively targets soluble vimentin in the glaucoma filtration surgical model of fibrosis.

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G0/G1 cell cycle inhibition (IC50 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27Kip1 expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27Kip1 expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis.

P21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

BackgroundHuman neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21Waf1 cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.MethodsVimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (n = 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (n = 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.ResultsWe extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.ConclusionOur results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.

Syncoilin modulates peripherin filament networks and is necessary for large-calibre motor neurons

Syncoilin is an atypical type III intermediate filament (IF) protein, which is expressed in muscle and is associated with the dystrophin-associated protein complex. Here, we show that syncoilin is expressed in both the central and peripheral nervous systems. Isoform Sync1 is dominant in the brain, but isoform Sync2 is dominant in the spinal cord and sciatic nerve. Peripherin is a type III IF protein that has been shown to colocalise and interact with syncoilin. Our analyses suggest that syncoilin might function to modulate formation of peripherin filament networks through binding to peripherin isoforms. Peripherin is associated with the disease amyotrophic lateral sclerosis (ALS), thus establishing a link between syncoilin and ALS. A neuronal analysis of the syncoilin-null mouse (Sync(-/-)) revealed a reduced ability in accelerating treadmill and rotarod tests. This phenotype might be attributable to the impaired function of extensor digitorum longus muscle and type IIb fibres caused by a shift from large- to small-calibre motor axons in the ventral root.

Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition

Vimentin is used widely as a marker of the epithelial to mesenchymal transitions (EMTs) that take place during embryogenesis and metastasis, yet the functional implications of the expression of this type III intermediate filament (IF) protein are poorly understood. Using form factor analysis and quantitative Western blotting of normal, metastatic, and vimentin-null cell lines, we show that the level of expression of vimentin IFs (VIFs) correlates with mesenchymal cell shape and motile behavior. The reorganization of VIFs caused by expressing a dominant-negative mutant or by silencing vimentin with shRNA (neither of which alter microtubule or microfilament assembly) causes mesenchymal cells to adopt epithelial shapes. Following the microinjection of vimentin or transfection with vimentin cDNA, epithelial cells rapidly adopt mesenchymal shapes coincident with VIF assembly. These shape transitions are accompanied by a loss of desmosomal contacts, an increase in cell motility, and a significant increase in focal adhesion dynamics. Our results demonstrate that VIFs play a predominant role in the changes in shape, adhesion, and motility that occur during the EMT.

Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis

We evaluated the expression of the type III intermediate filament (IF) protein, peripherin (PRP), in ubiquinated inclusions of motor neurons in amyotrophic lateral sclerosis (ALS). A previous study showed that overexpression of PRP in transgenic mice induces motor neuron disease with formation of PRP-containing inclusions before onset of symptoms [J. Cell Biol. 147 (3) (1999) 531]. To determine whether PRP inclusions occur in the human disease, we applied doublelabeling immunofluorescence to paraffin sections of the spinal cord obtained by autopsy of 40 ALS patients with sporadic disease and 39 controls. Inclusions that expressed immunoreactive ubiquitin and peripherin were recorded by video camera, and the sections were stained by hematoxylin and eosin (H&E) to define morphology. Lewy body-like inclusions (LBLIs) were seen in motor neuron perikarya of 9 of 40 ALS cases and none in controls; all LBLIs expressed peripherin. Skein-like inclusions (SLIs) were identified by ubiquitin, but did not express PRP with rare exceptions. Neither skein-like inclusions nor LBLIs expressed αB-crystallin, neurofilament protein (NF-L, NF-M and NF-H subunits), α-internexin, actin or α-synuclein. Immunoblot of the whole spinal cord exhibited a single 57-kDa band of peripherin in ALS patients and controls. Our data document the expression of peripherin in LBLIs, which may provide a clue to the pathogenesis of neurodegeneration in ALS.

Menin's Interaction with Glial Fibrillary Acidic Protein and Vimentin Suggests a Role for the Intermediate Filament Network in Regulating Menin Activity

Recently the multiple endocrine neoplasia type 1 ( MEN1) tumor suppressor gene was cloned. Its protein product, called menin, has been shown to associate with the AP1 transcription factor JunD and to repress JunD-mediated transcription. However, little is known concerning the regulation of menin. Here we report that menin interacts with the type III intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP) and vimentin. Menin's interaction with these IF proteins was characterized and confirmed both in vitro and in vivo using GST pull-down analysis, co-immunoprecipitation experiments, and immunofluorescence studies. Deletion mutants of GFAP or vimentin involving the head domains of the molecules abolish the interaction with menin. Endogenous menin is colocalized with GFAP and vimentin in glioma cells as determined by confocal microscopy. Furthermore, a tailless GFAP deletion mutant, which disrupts the IF network, results in menin/GFAP/vimentin-containing aggregates. Triple immunofluorescence labeling studies with antibodies against menin, BrdU, and GFAP show that menin and GFAP colocalize in glioma cells at the S–G2 phase of the cell cycle, as measured by BrdU incorporation. Our data suggest that the intermediate filament network interacts with and may serve as a cytoplasmic sequestering network for menin at the S and early G2 phase of the cell cycle.

Reduced number of unmyelinated sensory axons in peripherin null mice

Peripherin is a type III intermediate filament (IF) abundantly expressed in developing neurons, but in the adult, it is primarily found in neurons extending to the peripheral nervous system. It has been suggested that peripherin may play a role in axonal elongation and/or cytoskeletal stabilization during development and regeneration. To further clarify the function of peripherin, we generated and characterized mice with a targeted disruption of the peripherin gene. The peripherin null mice were viable, reproduced normally and did not exhibit overt phenotypes. Microscopic analysis revealed no gross morphological defects in the ventral and dorsal roots, spinal cord, retina and gut, but protein analyses showed increased levels of the type IV IF alpha-internexin in ventral roots of peripherin null mice. Whereas the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged in peripherin knockout mice, there was a substantial reduction ( approximately 34%) in the number of L5 unmyelinated sensory fibers that correlated with a decreased binding of the lectin IB4. These results demonstrate a requirement of peripherin for the proper development of a subset of sensory neurons.

Interaction in vitro of type III intermediate filament proteins with triplex DNA.

As previously shown, type III intermediate filaments (IFs) select from a mixture of linear mouse genomic DNA fragments mobile and repetitive, recombinogenic sequences that have also been identified in SDS-stable crosslinkage products of vimentin and DNA isolated from intact fibroblasts. Because these sequences also included homopurine.homopyrimidine (Pu.Py) tracts known to adopt triple-helical conformation under superhelical tension, and because IF proteins are single-stranded (ss) and supercoiled DNA-binding proteins, it was of interest whether they have a particular affinity for triplex DNA. To substantiate this, IF-selected DNA fragments harboring a (Pu.Py) segment and synthetic d(GA)(n) microsatellites were inserted into a vector plasmid and the constructs analyzed for their capacity to interact with IF proteins. Band shift assays revealed a substantially higher affinity of the IF proteins for the insert-containing plasmids than for the empty vector, with an activity decreasing in the order of vimentin > glial fibrillary acidic protein > desmin. In addition, footprint analyses performed with S1 nuclease, KMnO(4), and OsO(4)/bipyridine showed that the (Pu.Py) inserts had adopted triplex conformation under the superhelical strain of the plasmids, and that the IF proteins protected the triple-helical insert sequences from nucleolytic cleavage and chemical modification. All these activities were largely reduced in extent when analyzed on linearized plasmid DNAs. Because intramolecular triplexes (H-DNA) expose single-stranded loops, and the prokaryotic ssDNA-binding proteins g5p and g32p also protected at least the Pu-strand of the (Pu.Py) inserts from nucleolytic degradation, it seemed likely that the IF proteins take advantage of their ssDNA-binding activity in interacting with H-DNA. However, in contrast to g5p and E. coli SSB, they produced no clear band shifts with single-stranded d(GA)(20) and d(TC)(20), so that the interactions rather appear to occur via the duplex-triplex and triplex-loop junctions of H-DNA. On the other hand, the IF proteins, and also g32p, promoted the formation of intermolecular triplexes from the duplex d[A(GA)(20).(TC)(20)T] and d(GA)(20) and d(TC)(20) single strands, with preference of the Py (Pu.Py) triplex motif, substantiating an affinity of the proteins for the triplex structure as such. This triplex-stabilizing effect of IF proteins also applies to the H-DNA of (Pu.Py) insert-containing plasmids, as demonstrated by the preservation of intramolecular triplex-vimentin complexes upon linearization of their constituent supercoiled DNAs, in contrast to poor complex formation from free, linearized plasmid DNA and vimentin. Considering that (Pu.Py) sequences are found near MAR/replication origins, in upstream enhancer and promoter regions of genes, and in recombination hot spots, these results might point to roles of IF proteins in DNA replication, transcription, recombination, and repair.

Protein kinases required for segregation of vimentin filaments in mitotic process.

Vimentin, one of type III intermediate filament (IF) proteins, is expressed not only in mesenchymal cells but also in most types of tumor cells. In the present study, we introduced several types of vimentin mutated at putative phosphorylation sites in its amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation induced the formation of an unusually long bridge-like IF structure between the unseparated daughter cells, although these mutants formed the filament network similar to wild type in interphase cells. Together with sites phosphorylated by Rho-kinase and protein kinase C (PKC), vimentin-Ser72, which can not be phosphorylated by any known vimentin kinase, was one of the mutation sites essential for this phenotype. We further demonstrated that vimentin-Ser72 was phosphorylated specifically at the cleavage furrow during cytokinesis. These observations suggest the existence of a novel protein kinase responsible for vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. We propose that Rho-kinase, PKC, and an unidentified vimentin-Ser72 kinase may play important roles in vimentin filament separation during cytokinesis.

Late onset of motor neurons in mice overexpressing wild-type peripherin.

Peripherin, a type III intermediate filament (IF) protein, upregulated by injury and inflammatory cytokines, is a component of IF inclusion bodies associated with degenerating motor neurons in sporadic amyotrophic lateral sclerosis (ALS). We report here that sustained overexpression of wild-type peripherin in mice provokes massive and selective degeneration of motor axons during aging. Remarkably, the onset of peripherin-mediated disease was precipitated by a deficiency of neurofilament light (NF-L) protein, a phenomenon associated with sporadic ALS. In NF-L null mice, the overexpression of peripherin led to early- onset formation of IF inclusions and to the selective death of spinal motor neurons at 6 mo of age. We also report the formation of similar peripherin inclusions in presymptomatic transgenic mice expressing a mutant form of superoxide dismutase linked to ALS. Taken together, these results suggest that IF inclusions containing peripherin may play a contributory role in motor neuron disease.