Vimentin Human

What is the molecular structure of human vimentin and how does it contribute to its functions?

Human vimentin is a type III intermediate filament protein with a characteristic tripartite domain structure consisting of a central α-helical rod domain flanked by non-α-helical head and tail domains. The rod domain has been extensively characterized through crystallography and EPR spectroscopy, revealing a coiled-coil structure in segments 1B and 2, while segment 1A exhibits a single alpha helix rather than the expected coiled-coil structure .

The molecular structure of vimentin allows remarkable structural plasticity, enabling the protein to respond dynamically to mechanical stress. Using a "Divide and Conquer" approach, researchers have determined the crystal structure of several peptides derived from human vimentin . Recent advancements combine experimental EPR data with molecular modeling to complete the vimentin rod domain structure, particularly the previously unresolved L1-2 linker region (residues 248-263) .

Methodology for structural analysis typically involves:

• X-ray crystallography of vimentin peptides

• Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy

• Molecular dynamics (MD) simulations to validate models and understand dynamic behavior

• Construction of full-length models using molecular modeling software like MODELLER and UCSF CHIMERA

What is the expression pattern of vimentin across human tissues and how is it regulated?

Vimentin expression is remarkably widespread in human tissues. According to the Human Protein Atlas database, vimentin protein is expressed in the majority of the 44 tissues analyzed, with 14 tissues showing particularly high expression levels, including skin, lung, kidney, bone marrow, and lymph node . At the RNA level, vimentin is constitutively expressed across all major tissues as documented in the HPA, GTEx, and Fantom databases .

The regulation of vimentin expression involves multiple mechanisms:

• A serum response element in the VIM promoter responds to factors present in serum, explaining why many cell types express vimentin when cultured in serum-supplemented media

• During embryonic development, vimentin first appears in highly migratory cells when the embryo is still a two-layered epithelium and ectodermal cells begin migrating into the "mesodermal cleft"

• Postnatally, vimentin expression becomes restricted to fibroblasts, endothelial cells, lymphocytes, and specialized cells in the thymus and brain

• Upregulation occurs during epithelial-mesenchymal transition (EMT) in both cultured epithelial cells and in vivo contexts

Researchers should note that many cell types expressing vimentin in culture may not express it in vivo due to the serum effect, making them potentially problematic models for studying the genuine biological functions of vimentin .

What are the fundamental cellular functions of vimentin?

Vimentin plays critical roles in multiple cellular processes:

Cellular mechanics and structure:

• Maintains mechanical stability of cells

• Integrates mechanical stimuli from the environment

• Forms load-bearing superstructures that restrain F-actin retrograde flow

• Regulates cell cortical stiffness, which affects migration capacity

Cell migration and motility:

• Coordinates with focal adhesions and the actomyosin network to regulate cell motility

• Mediates bidirectional interactions with the actomyosin network that control cell migration

• Affects RhoA kinase signaling

• Modulates the dynamics of both microtubule and actomyosin networks

Additional cellular functions:

• Cell adhesion and wound healing

• Mechanosensing and mechanotransduction

• Cell senescence

• Transport of organelles and mRNA

• Regulation of protein synthesis

• Modulation of signaling pathways

How does vimentin influence cell migration on a molecular level?

Vimentin's role in cell migration involves complex molecular interactions with multiple cytoskeletal and signaling components:

Interaction with focal adhesion complexes:

• Vimentin regulates focal adhesion kinase (FAK), which imparts motile properties to cells

• By recruiting VAV2 (a Rac1 guanine nucleotide exchange factor) to FAK, vimentin regulates cell adhesion

• Vim−/− cells exhibit elevated levels of phosphorylated FAK and its targets, Src and ERK1/2

Coordination with actin dynamics:

• Vimentin intermediate filaments slow actin retrograde flow rates, which buffers traction stresses

• In Vim−/− cells, actin flows are more than an order of magnitude faster

• Vimentin restrains F-actin's retrograde flow and governs the alignment of traction stresses

• Influence on lamellipodium width, a key structure for pseudopodial migration

Mechanical effects:

• Cells lacking vimentin show significant decreases in cortical region stiffness

• This mechanical deficit contributes to impaired migratory activity and insufficient force generation for wound contraction

• Live-cell observations demonstrate that shear stress causes rapid deformation of vimentin networks, suggesting a role in mechanosensing and mechanotransduction

For studying these interactions, researchers employ:

• Traction force microscopy

• Sharp tip atomic force microscopy

• Time-lapse imaging of fluorescently tagged vimentin

• Comparative studies between wild-type and Vim−/− cells

What are the key phenotypes observed in vimentin knockout (Vim−/−) mouse models?

Vimentin knockout mice (Vim−/−) exhibit numerous phenotypes across different systems despite initially appearing to develop and reproduce normally. Key phenotypes include:

Vascular system abnormalities:

• Poor vascular development with fewer vessels and reduced branching

• Defects in arterial remodeling and vascular smooth muscle cell differentiation

• Elevated carotid stiffness, contractility, and endothelial dysfunction

• Failed vasodilation of the renal vascular system after partial nephrectomy, leading to renal failure

Wound healing and tissue repair defects:

• Impaired wound healing and contraction

• Defects in TGF-β signaling

• Impaired epithelial-to-mesenchymal transition

• Compromised vascularization during wound repair

Nervous system alterations:

• Hypermyelination of peripheral nerves

• Slower neurite outgrowth

• Delayed recovery of sensation following sciatic nerve crush

Metabolic and cellular abnormalities:

• Defects in lipid droplet organization

• Abnormalities in cell size regulation and mTORC1 signaling

• Decreased production of corticosterone affecting energy, immune reactions, and stress responses

Sex-specific effects:

• Increased mortality in male Vim−/− mice, but not in females

• Possibly related to higher susceptibility to environmental stress due to decreased corticosterone production

These diverse phenotypes highlight vimentin's critical roles across multiple physiological systems and provide valuable models for studying vimentin-related diseases.

How is vimentin implicated in cancer progression and metastasis?

Vimentin plays multifaceted roles in cancer progression:

EMT and cancer cell plasticity:

• Vimentin is a key marker for epithelial-mesenchymal transition (EMT), a critical process in tumor cell dissemination

• Experimental co-expression of vimentin and keratin in MCF-7 human breast cancer cells (MoVi clones) leads to phenotypic interconversion between epithelial and mesenchymal states

Enhanced invasion and motility:

• Overexpression of vimentin in MCF-7 cells leads to augmented motility and invasiveness in vitro

• These activities can be transiently down-regulated by vimentin antisense oligonucleotides

Proliferation and tumorigenicity:

Integrin profile alterations:

• Vimentin expression causes a decrease in α2- and α3-containing promiscuous integrins and β1-containing integrins

• Concurrent increase in α6-containing laminin receptor integrin

• Enhanced haptotactic migration toward laminin substrate

Cell surface vimentin:

• Vimentin can be expressed at the cell surface in addition to its cytoplasmic location

• Cell surface vimentin serves as a natural human immune response target

• Monoclonal antibodies reactive with cell surface vimentin have been obtained from sentinel lymph nodes of cancer patients

• Pritumumab, one such vimentin-reactive antibody, has been used to treat brain cancer patients

These findings suggest vimentin confers selective advantages to cancer cells in their interpretation of extracellular matrix signals, though vimentin alone may not be sufficient for metastasis.

What roles does vimentin play in inflammatory and autoimmune diseases?

Vimentin is implicated in several inflammatory and autoimmune conditions:

Rheumatoid Arthritis (RA):

• Biopsies from RA-affected tissues express fibrotic markers including vimentin, while healthy tissues express epithelial-like biomarkers

• Approximately 40% of sera from RA patients contain autoantibodies directed toward mutated citrullinated vimentin (MCV)

• Anti-MCV antibodies can be detected early in disease progression

• Anti-MCV titers correlate closely with disease progression, allowing for early diagnosis, prognosis, and therapeutic monitoring

• Citrullination of vimentin during inflammation triggers antigenic properties

Crohn's Disease:

• Associated with upregulation of vimentin protein levels

• Invasive properties of Crohn's disease cells are linked to vimentin expression

• Fibrotic areas show EMT-related markers, particularly vimentin, suggesting EMT involvement in pathogenesis

• Vimentin-targeted treatment of Crohn's-disease-associated Escherichia coli with withaferin-A promotes correct functioning of inflammatory response, autophagy, and cell invasion

Other Inflammatory Conditions:

• Vimentin serves as an important marker in many inflammatory conditions

• It plays roles in delayed wound healing

• Involved in cell surface binding and replication of viruses including HIV, SARS-CoV, dengue, and encephalitis viruses

• In HIV specifically, vimentin is associated with viral infectivity factor linked to HIV replication

The connection between vimentin and these conditions highlights its potential as both a diagnostic biomarker and therapeutic target.

What are the most effective methods for detecting and quantifying vimentin in experimental settings?

Researchers employ multiple techniques to assess vimentin:

Immunohistochemistry (IHC):

• Standard approach for tissue sections

• Can detect spatial distribution within tissues and cells

• Allows scoring of expression intensity and percentage of positive cells

• Permits co-localization studies with other markers

Immunofluorescence microscopy:

• Confocal immunofluorescence microscopy provides superior resolution

• Enables visualization of vimentin filament networks

• Particularly useful for studying dynamic changes in live cells

• Can be combined with computer-assisted imaging to quantify stained areas

Molecular techniques:

• Real-time quantitative RT-PCR (RT-qPCR) for mRNA expression

• Western blot for protein level detection and semi-quantification

• Flow cytometry for quantifying vimentin-positive cell populations

Advanced structural approaches:

• Site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy

• X-ray crystallography of vimentin peptides

• Molecular dynamics simulations to study structural dynamics

Live-cell imaging:

• Expression of fluorescently tagged vimentin

• Allows for real-time observation of network dynamics under various stimuli

• Particularly valuable for mechanosensing studies where rapid deformation can be visualized

When selecting a methodology, researchers should consider:

• The specific research question (expression level vs. spatial distribution vs. dynamics)

• Available tissue/sample type (fixed vs. fresh vs. cell culture)

• Need for quantitative vs. qualitative data

• Whether subcellular localization information is required

How can researchers effectively manipulate vimentin expression or function in experimental models?

Several approaches are available for modulating vimentin expression or function:

Genetic manipulation:

• Knockout models: Vim−/− mice are widely used to study systemic effects of vimentin loss

• siRNA/shRNA for transient or stable knockdown in cell culture models

• CRISPR-Cas9 genome editing for targeted modifications of the vimentin gene

• Transfection with vimentin expression vectors (as in MoVi clones) to study overexpression effects

Pharmacological interventions:

• Withaferin-A: A plant-derived inhibitor that binds to vimentin and disrupts its function

• Has been used in Crohn's disease models to target vimentin-mediated processes

• Other compounds that target vimentin assembly or stability

Post-translational modification targeting:

• Modulating citrullination of vimentin (relevant for autoimmune conditions)

• Targeting phosphorylation pathways that affect vimentin organization

Antibody-based approaches:

• Vimentin antisense oligonucleotides can transiently down-regulate vimentin-mediated activities

• Monoclonal antibodies against cell surface vimentin (like pritumumab) can be used for targeting vimentin-expressing cells

• Function-blocking antibodies can interfere with specific vimentin interactions

Experimental considerations:

• Cell type selection is crucial as many cultured cells express vimentin due to serum response elements in the VIM promoter

• Phenotypic changes may be context-dependent and influenced by compensatory mechanisms

• Temporal aspects are important as acute vs. chronic vimentin modulation may yield different results

What experimental approaches are most suitable for investigating vimentin's interactions with other cytoskeletal components?

Investigating vimentin's interactions with other cytoskeletal elements requires specialized techniques:

Colocalization studies:

• Dual or multi-color immunofluorescence microscopy

• Super-resolution microscopy techniques (STORM, PALM, SIM) for detailed spatial relationships

• Live-cell imaging with differently labeled cytoskeletal components

Biochemical interaction assays:

• Co-immunoprecipitation to identify protein-protein interactions

• Proximity ligation assays to detect close associations in situ

• Pull-down assays with purified components to test direct interactions

Functional perturbation approaches:

• Selective disruption of specific cytoskeletal elements (e.g., using cytochalasin D for actin, nocodazole for microtubules)

• Comparing effects in wild-type vs. Vim−/− cells

• Live imaging during cytoskeletal perturbations

Mechanical studies:

• Traction force microscopy to measure cell-generated forces

• Atomic force microscopy to assess local mechanical properties

• Optical tweezers or magnetic tweezers for direct mechanical manipulation

Computational and modeling approaches:

• Agent-based modeling of cytoskeletal interactions

• Molecular dynamics simulations to predict interaction interfaces

• Analysis of simultaneous dynamics of different cytoskeletal elements

Studies have revealed that vimentin interacts with both the actin and microtubule networks. For instance, research has shown that vimentin IFs slow actin retrograde flow rates, with actin flows being more than an order of magnitude faster in Vim−/− cells . These interactions are crucial for cell migration, as vimentin forms a load-bearing superstructure that restrains F-actin's retrograde flow and governs the alignment of traction stresses.

What is currently known about cell surface vimentin and its potential as an immunotherapeutic target?

Cell surface vimentin represents an emerging area of vimentin biology with significant therapeutic implications:

Expression and detection:

• Vimentin, typically considered a cytoplasmic protein, can also be expressed at the cell surface

• Several studies have confirmed this localization using surface biotinylation and non-permeabilized immunostaining approaches

• Expression has been detected on various cell types including cancer cells, activated macrophages, and platelets

Natural immune responses:

• Natural human monoclonal antibodies reactive with cell surface vimentin have been isolated from sentinel lymph nodes of cancer patients

• This suggests an innate auto-antigenic natural human immune response, potentially a function of immunosurveillance

• The recognition of cell surface vimentin may be part of a pre-established immune response to vimentin released during cell lysis

Therapeutic applications:

• Pritumumab, a vimentin-reactive antibody derived from lymph nodes of cancer patients, has been used to treat brain cancer patients

• The natural human origin of these antibodies may confer advantages for therapeutic applications

• Cell surface vimentin serves as an accessibility target compared to intracellular vimentin

Research questions under investigation:

• How does vimentin—and in what form—enter lymph nodes to stimulate immune responses?

• Is surface vimentin by itself, in an altered form, or complexed with other biomolecules?

• What role do post-translational modifications play in focusing a human response on particular vimentin epitopes?

• How is cell surface vimentin involved in epithelial-mesenchymal transition processes?

The study of cell surface vimentin opens new avenues for therapeutic approaches in cancer and potentially other vimentin-associated diseases.

How do post-translational modifications of vimentin affect its function in health and disease?

Post-translational modifications (PTMs) of vimentin critically influence its functions and disease associations:

Citrullination:

• Conversion of arginine residues to citrulline by peptidylarginine deiminases

• Citrullinated vimentin (MCV) becomes an autoantigen in rheumatoid arthritis

• Approximately 40% of RA patient sera contain anti-MCV antibodies

• These antibodies serve as valuable diagnostic and prognostic markers

• Citrullination during inflammation triggers antigenic properties within the filament

Phosphorylation:

• Regulates vimentin assembly/disassembly dynamics

• Multiple kinases target vimentin, including PKA, PKC, CDKs, and Rho kinase

• Phosphorylation states change during cell cycle progression, cellular stress, and migration

• Hyperphosphorylation can lead to filament reorganization or disassembly

Other modifications:

• Sumoylation: Affects vimentin solubility and assembly

• Glycosylation: May influence stability and interactions

• Oxidative modifications: Occur during cellular stress and aging

Methodological approaches to study PTMs:

• Mass spectrometry for comprehensive PTM mapping

• Phospho-specific or citrulline-specific antibodies

• Site-directed mutagenesis to create phosphomimetic or non-phosphorylatable variants

• Enzyme inhibitors to modulate specific modifications

Understanding vimentin PTMs provides insights into both normal regulatory mechanisms and pathological processes, particularly in contexts like autoimmunity where modified forms of vimentin become immunogenic.

What are the emerging therapeutic approaches targeting vimentin in disease contexts?

Vimentin represents a promising therapeutic target across several disease contexts:

Cancer therapy approaches:

• Monoclonal antibodies: Pritumumab and other antibodies targeting cell surface vimentin

• Small molecule inhibitors: Compounds disrupting vimentin assembly or stability

• Vimentin-targeted drug delivery: Exploiting cell surface vimentin for targeted delivery

• Gene therapy: Antisense oligonucleotides have shown efficacy in down-regulating vimentin-mediated activities in experimental models

Autoimmune disease strategies:

• Targeting citrullinated vimentin in rheumatoid arthritis

• Modulating immune responses to vimentin epitopes

• Blocking vimentin-mediated signaling in inflammatory contexts

Infectious disease interventions:

• Targeting vimentin's role as a receptor or co-receptor for viruses (HIV, SARS-CoV, dengue, encephalitis)

• Vimentin-targeted treatment of Crohn's-disease-associated bacteria with withaferin-A

Fibrosis and wound healing approaches:

• Modulating vimentin's role in EMT to control fibrotic processes

• Targeting vimentin-dependent TGF-β signaling

• Enhancing appropriate wound healing responses through vimentin-mediated pathways

Methodological considerations:

• Specificity challenges: Vimentin's widespread expression necessitates careful targeting

• Delivery strategies: Accessing intracellular vimentin vs. targeting cell surface vimentin

• Combination approaches: Vimentin-targeted therapies as part of multi-modal treatment strategies

How do we reconcile vimentin's diverse and sometimes contradictory functions in different cellular contexts?

The remarkable functional diversity of vimentin stems from several factors:

Structural adaptability:

• Vimentin intermediate filaments possess inherent structural plasticity

• The protein can form different assembly states (soluble, unit-length filaments, mature filaments)

• Dynamic reorganization occurs in response to cellular needs and stresses

• Molecular dynamics simulations show that the L1-2 linker region exhibits heterogeneity with concerted switching of states among dimer chains

Context-dependent interactions:

• Forms different protein complexes depending on cell type and physiological state

• Interacts with multiple cytoskeletal elements, including actin and microtubules

• Serves as a scaffold for signaling molecules in various pathways

• Functions both as a cytoplasmic and cell surface protein

Regulatory diversity:

• Subject to numerous post-translational modifications that alter function

• Expression levels vary significantly between tissues and developmental stages

• Different splice variants may predominate in different contexts

• Sex-specific effects suggest hormonal regulation (as seen in male vs. female Vim−/− mice)

Methodological considerations for researchers:

• Cell type selection is critical for relevance to in vivo function

• Awareness that serum in culture media induces vimentin expression in many cell types

• Need for complementary in vitro, cellular, and in vivo approaches

• Integration of structural, mechanical, and signaling perspectives

The apparent contradictions in vimentin function likely reflect its role as a cellular integrator—responding to and coordinating diverse inputs to maintain cellular homeostasis across varying conditions and requirements.