Recombinant Mouse Vezatin (Vezt)

What is Mouse Vezatin and what is its molecular structure?

Vezatin (Vezt) is a ubiquitous transmembrane protein that bridges myosin VIIA to the cadherin-catenins complex at adherens cell-cell junctions. Structurally, mouse Vezatin is characterized as an integral membrane protein with two adjacent transmembrane domains (approximately at amino acids 134-158 and 166-188), with both N-terminal and C-terminal regions facing the cytoplasm .

Two main mouse Vezatin isoforms have been identified:

• Long isoform (accession no. AAX12551): approximately 88 kDa

• Short isoform (accession no. AAX12552): approximately 71 kDa

These isoforms differ primarily in their C-terminal regions . Molecular evidence confirming Vezatin's transmembrane nature includes:

• Resistance to extraction with 0.1 M sodium carbonate (pH 11.3), which typically releases peripheral membrane proteins

• Recovery in plasma membrane fraction during ultracentrifugation experiments

• Detection in biotinylated cell surface protein fractions

Vezatin appears to form homodimers, as predicted by AlphaFold2 analyses, which has implications for its interactions with other protein complexes such as dynactin .

What methodologies are used to express and purify recombinant mouse Vezatin?

Successful expression and purification of recombinant mouse Vezatin typically involves:

Expression vectors:

• pCMV vector for N-terminal Myc-tagged Vezatin

• Modified pCS2 vector for C-terminal Myc-tagged Vezatin

• pFast Bac vector for Vezatin fragments (e.g., aa 188-607)

Expression systems:

• Mammalian cells (e.g., MDCKII) for full-length protein

• Bacterial systems (e.g., E. coli) for specific domains or fragments

• Insect cell expression systems for higher yields of properly folded protein

Purification strategies:

• For membrane-anchored Vezatin:

  • Detergent solubilization (e.g., using RIPA buffer containing 1% NP-40, 0.5% deoxycholate, 0.1% SDS)

  • Affinity chromatography using epitope tags

  • Size exclusion chromatography

• For truncated constructs:

  • ΔTM-VezA-GFP (deletion of transmembrane domains) can be used for pull-down experiments

  • Expression of specific domains for interaction studies

Validation methods:

• SDS-PAGE and western blotting using anti-Vezatin antibodies

• Functional binding assays with known partners (myosin VIIA, radixin, or dynactin components)

• Mass spectrometry for protein identification

Given the challenges of working with integral membrane proteins, many studies use truncated forms lacking the transmembrane domains to improve solubility and yields.

What are the primary functions of Vezatin in cellular adhesion and organization?

Vezatin plays several critical roles in cellular adhesion and organization:

At adherens junctions:

• Bridges myosin VIIA to the cadherin-catenins complex, linking adhesion molecules to the actin cytoskeleton

• Stabilizes mature adherens junctions rather than participating in their initial formation

• Co-localizes with E-cadherin at cell-cell contacts in various epithelia

Recruitment dynamics:

• Not detected at nascent cell-cell contacts where E-cadherin is already present

• Appears at junctions as they mature, forming discrete spots or continuous lines

• Double immunolabeling experiments show co-localization with E-cadherin specifically at adherens junctions, but not at desmosomes or focal adhesions

Protein interactions:

• Co-immunoprecipitates with E- or N-cadherin and β- and α-catenins

• Directly interacts with radixin in its actin-binding conformation

• Associates with the dynactin complex, affecting intracellular transport

Tissue-specific roles:

• Critical for sound resilience in cochlear hair cells

• Essential for dendritic spine morphogenesis in neurons

• Functions in facilitating dynactin complex assembly in various cell types

These functions collectively demonstrate Vezatin's importance in maintaining tissue integrity through strengthening cell-cell junctions and organizing cytoskeletal elements.

How is Vezatin expressed in different mouse tissues and what are its tissue-specific functions?

Vezatin demonstrates distinct expression patterns across multiple mouse tissues:

Immunofluorescence analysis confirms Vezatin's presence at cell-cell junctions in all these tissues, with tissue-specific localization patterns . Unlike some junction proteins, Vezatin shows specificity for adherens junctions, with no co-localization with desmoglein (a desmosome-specific protein) in the murine skin or intestine, and no detection at focal adhesion sites of fibroblasts .

The developmental increase in Vezatin expression at junctions between hair cells and supporting cells from postnatal day 4 to 16 correlates with maturation of the organ of Corti, suggesting developmentally regulated functions .

What are the molecular interactions between Vezatin and the cadherin-catenins complex?

Vezatin interacts with the cadherin-catenins complex through multiple molecular mechanisms:

Interaction evidence:

• Co-immunoprecipitation experiments show that Vezatin associates with E- or N-cadherin and β- and α-catenins in various cell types including MDCK cells, E- or N-cadherin transfected L cells, and S180 fibroblasts

• Biotinylation of cell surface proteins followed by streptavidin pull-down and immunoblotting confirms the association of Vezatin with E-cadherin at the plasma membrane

Temporal dynamics:

• Unlike E-cadherin, which is present at nascent contacts, Vezatin is recruited later to junctions

• In semi-confluent MDCK cultures, Vezatin shows punctate staining at initial cell-cell contacts, resembling E-cadherin-positive puncta identified as intermediates in adherens junction formation

• At cell confluence, both Vezatin and E-cadherin become broadly distributed along the adherens junction

Functional significance:

• While weak cell-cell adhesion is obtained by homophilic interaction of the cadherin ectodomain, strong adhesion requires linking to the actin cytoskeleton

• Vezatin likely strengthens this linkage by connecting the cadherin-catenin complex to myosin VIIA and thereby to the actin cytoskeleton

• The delayed recruitment pattern suggests Vezatin functions in stabilizing rather than establishing junctions

These interactions position Vezatin as a critical component for maintaining robust adherens junctions by reinforcing the connection between adhesion receptors and the cytoskeleton.

What phenotypes are observed in Vezatin knockout mouse models?

Conditional Vezatin knockout mice exhibit distinct phenotypes depending on the tissue-specific deletion:

Inner ear-specific knockouts (Vezt^flox/flox:PrestinCre):

• Increased susceptibility to noise-induced hearing loss (irreversible hearing loss after only one minute exposure to 105 dB broadband sound)

• Spontaneous late onset progressive hearing loss

• Vestibular dysfunction

• Substantial hair cell death

Neuron-specific knockouts (Vezt^flox/flox:CamKII-Cre):

• Defects in dendritic spine morphogenesis

• Altered spine density

• Changes in synaptic function

In vitro Vezatin knockdown in neurons:

• Quantification of Vezatin-positive spines on a similar dendritic length in knockdown (Vezt^fl/fl:LV-Cre) and control (Vezt^+/+:LV-Cre) neurons shows significant reduction in Vezatin-positive structures

• Reduced spine formation or stability

Methodology for phenotypic characterization:

• RT-PCR analysis to confirm deletion of targeted Vezt exons

• Immunohistochemistry to verify protein loss

• Functional testing (e.g., hearing tests)

• Microscopic analysis of tissue structure and integrity

• Quantitative analysis of cellular structures (spines, hair cells)

These models demonstrate that Vezatin is essential for maintaining structural integrity and function in specialized tissues, particularly those subject to mechanical stress or requiring precise cellular architecture.

How does Vezatin contribute to hearing function and protection against noise-induced hearing loss?

Vezatin plays a critical role in maintaining hearing function and protecting against noise-induced damage:

Expression pattern in the inner ear:

• Vezatin immunoreactivity at junctions between hair cells and supporting cells increases during postnatal development (P4 to P16)

• This coincides with the maturation of the organ of Corti and establishment of hearing function

Functional evidence from conditional knockout models:

• Vezatin^flox/flox:PrestinCre mice (with hair cell-specific deletion) show:

  • Irreversible hearing loss after only one minute of exposure to 105 dB broadband sound

  • Spontaneous progressive hearing loss even without noise exposure

  • Vestibular dysfunction indicating broader inner ear defects

Molecular mechanisms of protection:

• Vezatin strengthens adherens junctions between hair cells and supporting cells

• These reinforced junctions likely provide mechanical resilience against sound-induced vibrations

• The connection to myosin VIIA may be particularly important, as mutations in myosin VIIA also cause hearing loss

• Interaction with radixin in its actin-binding conformation suggests a role in organizing the actin cytoskeleton critical for hair cell function

Structural consequences of Vezatin deficiency:

• Substantial hair cell death observed in knockout models

• Compromised junctional integrity likely leads to mechanical damage during sound exposure

• Progressive deterioration of sensory epithelium structure over time

These findings identify Vezatin as a potential therapeutic target for noise-induced hearing loss and suggest that strengthening adherens junctions could protect against acoustic trauma.

What techniques are used to study Vezatin's interactions with binding partners?

Researchers employ multiple complementary techniques to characterize Vezatin's protein-protein interactions:

In vitro binding assays:

• Biotinylated fragments (e.g., A34 peptide, amino acids 336-569) used to test binding to myosin VIIA domains

• Recombinant proteins used to map interaction domains (e.g., C-terminal FERM domain of myosin VIIA)

• GST-fusion proteins of radixin domains (FERM domain, residues 1-310; C-terminal domain, residues 476-583) to map interaction sites

Pull-down experiments:

• ΔTM-VezA-GFP (Vezatin lacking transmembrane domains) can pull down interaction partners from cell extracts

• Arp11-GFP pull-downs to assess dynactin complex interactions

• Analysis of pulled-down proteins by mass spectrometry or immunoblotting

Co-immunoprecipitation:

• Anti-vezatin antibodies used to co-immunoprecipitate binding partners from cell extracts

• Detection of associated proteins (E-cadherin, catenins, myosin VIIA) by western blotting

Mutational analysis:

• Site-directed mutagenesis (e.g., T564A in radixin) to test functional importance of specific residues

• Creation of deletion mutants (e.g., vezAΔ1-20-GFP and vezAΔ563-615-GFP) to map interaction domains

Structural prediction:

• AlphaFold2 used to model potential direct interactions between Vezatin and partners

• Prediction that Vezatin forms homodimers

• Modeling of the N-terminus (aa 1-20) interaction with dynactin pointed end via p62 and p25

Cellular localization:

• Double immunofluorescence to detect co-localization with binding partners

• Live-cell imaging of fluorescently tagged proteins to study dynamics

These diverse approaches collectively provide robust evidence for specific interactions and help map the functional domains involved.

What is the role of Vezatin in dynactin complex assembly and intracellular transport?

Recent research has revealed that Vezatin (VezA) plays a crucial role in facilitating proper assembly of the dynactin complex, which is essential for dynein-mediated intracellular transport:

Functional impact on dynein-dynactin system:

• VezA affects microtubule plus-end accumulation of dynein before cargo binding

• Influences cargo adapter-mediated dynein activation

• Both processes require intact dynactin

Molecular interactions with dynactin components:

• VezA physically interacts with dynactin via the Arp1 mini-filament and its pointed-end sub-complex

• AlphaFold2 predictions suggest VezA's N-terminus (aa 1-20) interacts with the pointed end via p62 and p25

• VezA's C-terminal α-helix (563-615) docks in a pocket formed by two Arp1 subunits

Assembly mechanism:

• VezA doesn't affect assembly of the pointed-end sub-complex itself

• Instead, facilitates the connection between the Arp1 mini-filament and its pointed-end sub-complex

• In VezA-deficient cells, Arp11-GFP pulls down normal amounts of p25 and p62 but much lower amounts of Arp1, p50, p150, and capping protein

Experimental evidence:

• Conditional mutations in dynactin components show that assembly must be highly coordinated

• Loss of VezA causes partial defects in dynein-mediated transport processes

• Most notably affects early endosome distribution, with abnormal accumulation at hyphal tips in fungal models

Evolutionary conservation:

• This function appears conserved from fungi to mammals

• Mammalian vezatin associates with Arp1 in human interactome studies

• Vezatin is in close proximity to Arp1 as revealed by BioID proximity labeling

This role in dynactin assembly represents a novel function of Vezatin distinct from but complementary to its established role at adherens junctions, highlighting its versatility in organizing cellular structures.

What role does Vezatin play in dendritic spine morphogenesis and neuronal function?

Vezatin has emerged as an essential factor in dendritic spine development and neuronal connectivity:

Neuronal localization:

• Vezatin-GFP localizes to dendritic spines in cultured neurons

• Co-localizes with RFP-actin, suggesting involvement in actin cytoskeleton organization within spines

Experimental approaches for studying neuronal Vezatin:

• Dissociated neurons from rat cortex cultured and transfected at 7 DIV using LipofectAMINE 2000

• Time-lapse imaging experiments to monitor spine dynamics

• Lentiviral vectors (LV-Cre) used for in vitro knockdown in floxed Vezatin neurons

• Immunostaining with antibodies against Vezatin, Cre recombinase, and MAP2 (to visualize dendritic trees)

Functional consequences of Vezatin deficiency:

• Conditional knockout or knockdown results in altered spine morphology

• Quantification shows reduced number of Vezatin-positive spines

• Changes likely affect synaptic transmission and neuronal connectivity

Molecular mechanisms:

• Similar to its role in adherens junctions, Vezatin likely connects adhesion molecules at synapses to the actin cytoskeleton

• Interaction with radixin in its actin-binding conformation suggests a role in organizing synaptic actin

• May stabilize interactions between pre- and post-synaptic membranes

Implications for neurological disorders:

• Given Vezatin's role in spine morphogenesis, its dysfunction could potentially contribute to conditions characterized by synaptic abnormalities

• Understanding Vezatin's neuronal functions may provide insights into developmental and degenerative neurological conditions

These findings extend our understanding of Vezatin beyond classical epithelial junctions to specialized neuronal structures, highlighting its versatility in organizing cellular adhesion and cytoskeletal elements across diverse tissues.

How do researchers validate the functional significance of recombinant Vezatin in experimental systems?

Validating the functional significance of recombinant Vezatin requires multiple complementary approaches:

Expression validation:

• Western blotting to confirm expression of recombinant protein at expected molecular weight

• Immunofluorescence to verify proper subcellular localization

• Co-localization with known binding partners (e.g., E-cadherin at adherens junctions)

Functionality assays:

• Binding partner interactions:

  • In vitro binding assays with known partners (myosin VIIA, radixin, dynactin components)

  • Co-immunoprecipitation to confirm interaction with the cadherin-catenins complex

  • Pull-down experiments followed by mass spectrometry

• Rescue experiments:

  • Expression of recombinant Vezatin in Vezatin-deficient cells or tissues

  • Assessment of phenotypic rescue (e.g., junction formation, spine morphology)

  • Comparison of wild-type versus mutated forms to identify functional domains

• Live-cell imaging:

  • Time-lapse microscopy of cells expressing fluorescently tagged Vezatin

  • Analysis of recruitment dynamics to newly forming junctions

  • Co-imaging with other tagged proteins (e.g., RFP-actin) to study co-localization

• Structure-function analyses:

  • Creation of domain deletion mutants (e.g., ΔTM-VezA-GFP, vezAΔ1-20-GFP, vezAΔ563-615-GFP)

  • Expression of specific domains to identify minimal functional units

  • Site-directed mutagenesis of key residues predicted to mediate interactions

Controls for specificity:

• Comparison with empty vector or irrelevant protein expression

• Use of related but functionally distinct proteins

• Antibody validation in Vezatin knockout tissues

These approaches collectively ensure that recombinant Vezatin faithfully recapitulates the functions of the native protein and help delineate the structural basis of its diverse cellular roles.

What are the latest research findings on the role of Vezatin in different cellular pathways?

Recent research has expanded our understanding of Vezatin's roles beyond its classical function at adherens junctions:

1. Dynactin complex assembly:

• VezA/vezatin facilitates proper assembly of the dynactin complex

• Affects the connection between Arp1 mini-filament and its pointed-end sub-complex

• Impacts dynein-mediated intracellular transport of various cargoes

• This function appears evolutionarily conserved from fungi to mammals

2. Neuronal development and function:

• Essential for dendritic spine morphogenesis

• Localizes to dendritic spines and co-localizes with actin

• Conditional knockout affects spine formation and potentially synaptic function

• May link synaptic adhesion molecules to the actin cytoskeleton

3. Inner ear function and hearing protection:

• Critical for sound resilience of cochlear hair cells

• Mice lacking Vezatin in hair cells show extreme sensitivity to noise exposure

• Also develop spontaneous progressive hearing loss and vestibular dysfunction

• Suggests a role in maintaining mechanical integrity of the sensory epithelium

4. Mechanistic insights into protein interactions:

• Direct interaction with radixin in its actin-binding conformation

• N-terminus (aa 1-20) interaction with dynactin pointed end via p62 and p25

• C-terminal α-helix (563-615) docks in a pocket formed by two Arp1 subunits

• Vezatin likely forms homodimers, adding complexity to its interaction network

5. Potential relevance to disease mechanisms:

• Given its roles in tissue integrity, hearing protection, and neuronal connectivity

• May have implications for understanding disorders involving cellular adhesion defects

• Could represent a therapeutic target for noise-induced hearing loss or related conditions

These diverse findings collectively position Vezatin as a multifunctional protein with important roles in cellular architecture, mechanical resilience, and intracellular organization across various tissues and cell types.

What challenges exist in working with recombinant Vezatin and how can they be overcome?

Working with recombinant Vezatin presents several technical challenges that researchers must address:

Membrane protein expression obstacles:

• As an integral membrane protein with two transmembrane domains, Vezatin is difficult to express in soluble form

• Hydrophobic domains often cause protein aggregation or misfolding

• The large size (~88 kDa for long isoform) adds further complexity

Expression system limitations:

• Bacterial systems lack appropriate post-translational modifications and membrane folding machinery

• Mammalian systems provide better folding but lower yields

• Insect cell systems represent a compromise but may not recapitulate all modifications

Solutions and workarounds:

• Use of truncated constructs:

  • ΔTM-VezA-GFP (lacking transmembrane domains) for pull-down experiments

  • Expression of specific domains for interaction studies (e.g., aa 188-607)

  • Creation of minimal functional fragments for structural studies

• Optimization of expression conditions:

  • Careful selection of detergents for solubilization (e.g., RIPA buffer components)

  • Temperature modulation during expression

  • Use of specialized host strains or cell lines

• Advanced purification strategies:

  • Tandem affinity tags to improve purity

  • Size exclusion chromatography to separate aggregates

  • On-column refolding for bacterial expression

• Alternative approaches:

  • Cell-free expression systems

  • Nanodiscs or liposomes to provide membrane environment

  • Peptide mimetics of key interaction domains

• Validation methods:

  • Functional binding assays with known partners

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to verify proper folding

These strategies have enabled researchers to overcome the inherent difficulties in working with transmembrane proteins like Vezatin and to gain insights into its structure and function despite these challenges.

How can researchers study the interaction between Vezatin and the cytoskeleton?

Investigating Vezatin's interactions with cytoskeletal components requires specialized approaches:

Direct binding studies:

• In vitro binding assays using purified components:

  • Recombinant Vezatin (or domains) with purified actin

  • Interactions with actin-binding proteins (e.g., radixin, myosin VIIA)

  • GST-fusion proteins of radixin domains (FERM domain, residues 1-310; C-terminal domain, residues 476-583)

Co-localization analyses:

• Double or triple immunofluorescence staining for:

  • Vezatin and actin filaments

  • Vezatin and myosin VIIA

  • Vezatin and radixin

• Advanced microscopy techniques:

  • Super-resolution microscopy to precisely localize components

  • FRET or BRET to assess proximity of interactions

Live-cell imaging approaches:

• Co-transfection of cells with:

  • Vezatin-GFP and monomeric RFP-actin

  • Other fluorescently tagged cytoskeletal components

• Time-lapse imaging to study dynamics of interactions

• Analysis using inverted microscope with appropriate fluorescence capabilities

Functional perturbation experiments:

• Expression of dominant-negative Vezatin fragments

• Site-directed mutagenesis of key interaction residues

• Treatment with cytoskeleton-disrupting drugs

• Assessment of effects on:

  • Adherens junction stability

  • Dendritic spine morphology

  • Intracellular transport processes

Biochemical approaches:

• Actin co-sedimentation assays

• Cytoskeleton fractionation followed by immunoblotting

• Cross-linking studies to capture transient interactions

• Pull-down of cytoskeletal components using recombinant Vezatin domains

These methodologies collectively provide a comprehensive toolkit for dissecting the complex relationships between Vezatin and various cytoskeletal elements in different cellular contexts.

What is known about the developmental regulation of Vezatin expression?

The developmental regulation of Vezatin expression shows tissue-specific and temporally controlled patterns:

Inner ear development:

• Vezatin immunoreactivity at junctions between hair cells and supporting cells increases from postnatal day 4 (P4) to P16

• This coincides with the maturation of the organ of Corti and establishment of hearing function

• The developmental timing suggests a role in the functional maturation of auditory sensory epithelium

Early embryonic expression:

• Two main mouse vezatin isoforms (long: ~88 kDa and short: ~71 kDa) have been detected in mouse pre-implantation embryos

• These isoforms differ primarily in their C-terminal regions

• May play roles in establishing and maintaining early embryonic cell adhesion

Nervous system development:

• Expression in neurons correlates with periods of synaptogenesis and spine formation

• Conditional knockout in neurons using CamKII-Cre affects spine morphogenesis

• Suggests developmental roles in establishing neuronal connectivity

Methodological approaches to study developmental expression:

• RT-PCR analysis to detect vezatin transcripts at different developmental stages

• Immunohistochemistry to visualize protein localization during development

• Western blotting to quantify protein levels across developmental time points

• Creation of developmental stage-specific conditional knockout models

Regulation mechanisms:

• Transcriptional control during tissue differentiation

• Post-transcriptional regulation affecting isoform expression

• Post-translational modifications potentially modulating protein function

• Recruitment to specific subcellular locations during junction maturation

Understanding the developmental regulation of Vezatin expression provides insights into its roles in tissue morphogenesis, maturation, and maintenance across different organ systems.