Vimentin Bovine

What is vimentin and what are its primary functions in bovine cells?

Vimentin is the major protein subunit of 10nm or intermediate filaments (IFs) found in various cell types of mesenchymal and epithelial origin. In bovine systems, vimentin plays critical roles in maintaining cellular structure, facilitating adhesion and migration, and participating in signal transduction pathways. Vimentin filaments form complex networks that undergo constant reorganization, with approximately 8% of vimentin intermediate filaments (VIFs) exhibiting directed microtubule-based motion regardless of their location within the cell . The protein is particularly important in developmental processes, as evidenced by its expression during epiblast formation in bovine embryos . Vimentin also forms copolymers with other IFs such as GFAP (in astrocytes), desmin (in muscle cells), and neurofilament proteins (in developing neurons) .

What are the established methods for isolating and purifying vimentin from bovine tissues?

Several approaches have been validated for isolating bovine vimentin:

• Recombinant Expression System: The most consistent method involves using E. coli to express recombinant bovine vimentin. This approach has been successfully employed to produce purified human vimentin protein, with similar protocols applicable to bovine vimentin .

• Differential Extraction Method:

  • Homogenize bovine tissue in low-salt buffer (typically 10mM Tris-HCl, pH 7.4)

  • Remove cellular debris through centrifugation (10,000×g)

  • Pellet intermediate filaments using high-speed centrifugation (100,000×g)

  • Solubilize vimentin using high-salt buffer with detergent

  • Purify through column chromatography

• Immunoprecipitation: For analytical purposes, vimentin can be immunoprecipitated using validated antibodies and protein A-Sepharose beads. One validated protocol involves incubating cell lysate (pH 7.4) with protein A-Sepharose beads for 1 hour at 4°C, followed by washing and elution steps .

Verification of purified vimentin typically involves Western blotting with specific antibodies, with bovine vimentin appearing at the expected molecular weight of approximately 50 kDa .

What are the optimal immunodetection approaches for studying vimentin in bovine cells?

Multiple complementary techniques can be employed for robust vimentin detection in bovine systems:

For optimal results, researchers should validate antibodies specifically for bovine vimentin, as epitope availability can vary between species. The mouse monoclonal antibody clone 2D1 has been validated for detecting bovine vimentin in multiple applications . Proper controls, including isotype-matched control antibodies (e.g., IgG1 at 1:200 dilution), are essential to distinguish specific from non-specific binding .

How can researchers track dynamic changes in vimentin organization in living bovine cells?

Studying vimentin dynamics in live bovine cells requires specialized techniques:

• Fluorescent Protein Tagging:

  • Transfect bovine cells with vimentin-GFP/RFP fusion constructs

  • Allow for incorporation into endogenous networks

  • Use time-lapse confocal microscopy to track movement and reorganization

• Single-Particle Tracking:

  • The sparse vimentin-SunTag labeling strategy enables tracking of individual vimentin filaments even within dense networks

  • This technique has revealed that individual VIFs within bundles move independently of one another

  • Studies using this approach have demonstrated that approximately 8% of vimentin filaments undergo directed movement at any given time

• High-Resolution 3D Imaging:

  • Focused ion beam scanning electron microscopy (FIB-SEM) of vitrified cells allows three-dimensional reconstruction of vimentin networks

  • This technique has revealed that vimentin bundles form loosely organized, semi-coherent structures rather than tightly cross-linked assemblies

When applying these techniques to bovine cells, researchers should optimize transfection protocols specific to the bovine cell type being studied, as primary bovine cells often show lower transfection efficiencies compared to established cell lines.

What methodological approaches can distinguish between different vimentin states in bovine cells?

Vimentin exists in multiple states within bovine cells, requiring specific methods for differentiation:

• Soluble vs. Filamentous Vimentin:

  • Differential extraction with varying detergent and salt concentrations

  • Low-salt buffers maintain filamentous structure while high-salt/detergent buffers solubilize vimentin

  • Western blotting of fractions reveals distribution between states

• Phosphorylated vs. Non-phosphorylated Forms:

  • Phospho-specific antibodies detect specific modified residues

  • Phosphatase treatment as a control to confirm specificity

  • 2D gel electrophoresis separates phosphorylated isoforms

• Cell Surface vs. Cytoplasmic Vimentin:

  • Surface biotinylation followed by immunoprecipitation identifies cell-surface vimentin

  • Comparison of permeabilized vs. non-permeabilized immunostaining

  • Flow cytometry on live cells detects only surface-expressed protein

• Copolymers vs. Homopolymers:

  • Co-immunoprecipitation with antibodies against partner IFs (GFAP, desmin)

  • Double immunofluorescence to examine colocalization patterns

  • Proximity ligation assays detect close associations between different IF proteins

These approaches provide comprehensive analysis of vimentin's diverse functional states within bovine cellular systems.

How does vimentin expression correlate with embryonic developmental competence in bovine systems?

Vimentin expression serves as a valuable marker for embryo quality and developmental potential in bovine systems:

Research has demonstrated that vimentin transcript levels in day 8 blastocysts correlate significantly with developmental competence. Fast-developing bovine embryos (those expanded by 168 hours post-insemination) show significantly higher vimentin transcript levels compared to slower-developing counterparts . This temporal pattern continues through development:

The progressive increase in vimentin expression corresponds with critical developmental transitions, particularly inner cell mass differentiation into epiblast and hypoblast lineages. Vimentin protein localizes as filaments within specific portions of the ICM, indicating its role as a potential lineage marker during bovine embryonic development .

For researchers evaluating embryo quality, these findings suggest that vimentin expression analysis, particularly at the transcript level on day 8, may serve as a predictor of developmental potential in bovine embryos produced through in vitro fertilization procedures.

What methodological approaches can effectively analyze vimentin's role in bovine cell differentiation?

To investigate vimentin's function during bovine cell differentiation, researchers can employ several complementary strategies:

• Loss-of-Function Studies:

  • RNA interference (siRNA or shRNA) targeting bovine vimentin

  • CRISPR/Cas9-mediated knockout in bovine cell lines

  • Dominant-negative vimentin mutant expression

• Lineage Tracing with Vimentin Reporters:

  • Vimentin promoter-driven fluorescent reporters

  • Time-lapse imaging during differentiation processes

  • Correlation of vimentin expression with lineage-specific markers

• Multi-parameter Analysis of Differentiation:

  • Combined analysis of vimentin with other cytoskeletal markers

  • Flow cytometry to quantify changes across populations

  • Single-cell transcriptomics to identify co-regulated genes

• Morphological Assessment Methods:

  • Quantitative image analysis of vimentin network architecture

  • Correlation between network organization and differentiation state

  • 3D reconstruction of vimentin filament systems during differentiation

For bovine mammary epithelial cell differentiation studies, researchers have successfully employed real-time PCR, flow cytometry, and immunofluorescence microscopy to characterize vimentin expression . These studies revealed distinct vimentin expression patterns between primary and immortalized bovine mammary epithelial cell cultures, providing insights into their differentiation states.

How does vimentin interact with other cytoskeletal and adhesion components in bovine cells?

Vimentin establishes complex interactions with multiple cellular components in bovine systems:

• Vimentin-Microtubule Interactions:

  • Vimentin filaments utilize microtubule-based transport for movement throughout the cell

  • Approximately 8% of vimentin filaments undergo directed microtubule-based motion

  • Individual vimentin filaments within bundles can move independently, emerging from semi-coherent structures to engage with nearby microtubules

• Vimentin-Integrin Crosstalk:

  • Vimentin associates with focal adhesions containing integrins

  • Studies in mammalian cells have shown that vimentin filaments extend into lamellipodia in approximately 80% of control cells

  • This extension is reduced when β4-integrin is knocked down, suggesting coordination between adhesion systems and vimentin organization

  • Complexes of α6β4 integrin and vimentin act as signaling hubs

• Interaction with Other Intermediate Filaments:

  • Vimentin forms copolymers with other IFs including GFAP (in astrocytic cells), desmin (in muscle cells), and neurofilament proteins (in developing neurons)

  • These heteropolymers exhibit unique mechanical properties compared to homopolymers

• Vimentin and Focal Adhesions:

  • Knockdown of vimentin affects focal adhesion density without changing focal adhesion area

  • This effect is likely indirect, as direct focal adhesion-vimentin association is rare (approximately 3% of focal adhesions)

These interactions create a dynamic cytoskeletal network that responds to cellular needs during development, differentiation, and migration in bovine cells.

What are common pitfalls in immunodetection of bovine vimentin, and how can they be avoided?

Researchers commonly encounter several challenges when detecting bovine vimentin:

• Cross-Reactivity Issues:

  • Problem: Some anti-vimentin antibodies show cross-reactivity with other intermediate filament proteins.

  • Solution: Validate antibody specificity using vimentin-knockout controls or competitive binding assays. Select antibodies specifically validated for bovine vimentin, such as mouse monoclonal antibodies that recognize conserved epitopes .

• Fixation Artifacts:

  • Problem: Different fixation methods can dramatically alter vimentin staining patterns.

  • Solution: For immunocytochemistry, methanol fixation (100% v/v at -20°C for 10 minutes) generally preserves vimentin filament structure better than formaldehyde-based fixatives . Compare multiple fixation protocols for optimal results.

• Epitope Masking:

  • Problem: Post-translational modifications can mask vimentin epitopes.

  • Solution: Test multiple antibodies targeting different regions of vimentin. Consider antigen retrieval methods if working with fixed tissues.

• False Negatives in Flow Cytometry:

  • Problem: Inadequate permeabilization leads to poor vimentin detection.

  • Solution: Optimize permeabilization conditions; methanol permeabilization (100% v/v, -20°C, 10 minutes) has been validated for bovine cell analysis .

• Background and Specificity:

  • Problem: High background staining in immunodetection techniques.

  • Solution: Include proper blocking (e.g., dPBS containing 2% w/v BSA and 4% v/v goat serum for 1 hour) and use appropriate isotype controls (e.g., IgG1 at 1:200 dilution) to distinguish specific from non-specific binding .

For all immunodetection approaches, validation with multiple methods provides greater confidence in the specificity of vimentin detection in bovine systems.

How can researchers resolve contradictory data regarding vimentin function in bovine systems?

When encountering contradictory results in vimentin research:

• Evaluate Cell Type-Specific Differences:

  • Vimentin functions can vary significantly between cell types

  • For example, the relationship between vimentin and β4 integrin differs between lung cancer cells and breast cancer cells

  • Document precise cell types and compare only equivalent cell populations

• Consider Developmental Stage Variations:

  • Vimentin expression changes dramatically during development

  • In bovine blastocysts, significant differences exist between day 8, 9, and 12 embryos

  • Ensure temporal comparisons are appropriate when integrating data from different studies

• Assess Methodology Differences:

  • Detection techniques vary in sensitivity and specificity

  • For example, vimentin transcript detection has proven more sensitive than protein detection in early bovine embryos

  • Compare experimental approaches (WB, ICC, RT-PCR) when evaluating seemingly contradictory findings

• Consider Context-Dependent Functions:

  • In some contexts, vimentin primarily provides structural support

  • In others, it serves as a signaling hub or regulator of cellular processes

  • The dynamic nature of vimentin bundles, where individual filaments move independently within loosely organized structures , may explain apparently contradictory observations

Systematic documentation of all experimental variables and seeking patterns that might explain divergent findings is essential for reconciling contradictory data in vimentin research.

What are the most sensitive methods for detecting low levels of vimentin in bovine samples?

For detecting minimal vimentin expression in bovine systems:

• Quantitative PCR:

  • Can detect vimentin transcripts when protein is below detection threshold

  • Has successfully identified vimentin expression in day 8 bovine blastocysts when immunostaining was negative

  • Requires proper housekeeping gene selection for bovine systems

• Nested PCR:

  • Provides enhanced sensitivity for low-abundance transcripts

  • Particularly useful for single embryo or limited sample analysis

  • Requires careful control for contamination

• Immunoprecipitation Followed by Western Blotting:

  • Concentrates vimentin from dilute samples

  • Protocol using protein A-Sepharose beads with cell lysate (pH 7.4) incubated for 1 hour at 4°C has been validated

  • Enhances detection sensitivity compared to direct Western blotting

• Tyramide Signal Amplification (TSA):

  • Enhances immunofluorescence sensitivity by enzymatic signal amplification

  • Can detect proteins present at very low copy numbers

  • Requires careful optimization to prevent background issues

• Proximity Ligation Assay (PLA):

  • Detects protein-protein interactions involving vimentin

  • Each interaction generates a distinct fluorescent spot

  • Provides spatial information about low-abundance interactions

For developmental studies in bovine embryos, combining transcript analysis (qPCR) with optimized protein detection methods provides the most comprehensive assessment of vimentin expression dynamics.