VTA1 Antibody

What is VTA1 and why is it important in cellular biology?

VTA1 is a 34 kDa protein (307 amino acids) involved in the endosomal multivesicular bodies pathway. It serves as a cofactor of VPS4A/B, which catalyzes the disassembly of membrane-associated ESCRT-III assemblies. VTA1 is critical for membrane protein degradation, including stimulated growth factor receptors and lysosomal enzymes . Additionally, VTA1 plays significant roles in HIV-1 budding and has recently been identified as having unique regulatory functions in cytokinesis through interaction with the abscission checkpoint complex . The protein has alternative names including C6orf55, HSPC228, DRG-1, LIP5, and SBP1, reflecting its discovery in different research contexts .

What are the key considerations when selecting a VTA1 antibody?

When selecting a VTA1 antibody for research, consider these critical factors:

The antibody's validation data in your specific experimental system is particularly important, as reactivity may vary across different cell types and tissues.

What is the structure and domain organization of VTA1?

VTA1 possesses a modular structure with two well-folded terminal domains connected by a disordered middle linker region . The N-terminal domain (Vta1NTD) contains two MIT (microtubule interacting and transport) motifs similar to those found in Vps4, which form surface grooves that mediate interactions with accessory ESCRT-III proteins Vps60 and Vps46/Did2 . Key residues in these interactions include Trp-122 and Lys-152 . The C-terminal domain mediates VTA1 dimerization, which is necessary for its function as a Vps4 regulator . Both subunits in the VTA1 dimer are required for proper regulation of Vps4 activity. Understanding this domain organization is crucial when selecting antibodies that target specific epitopes.

What is the optimal protocol for Western blot using VTA1 antibody?

For optimal Western blot detection of VTA1, follow this protocol:

• Sample preparation: Prepare cell or tissue lysates in a compatible lysis buffer (e.g., NETN buffer)

• Protein separation: Use 12% SDS-PAGE for optimal resolution of the 34 kDa VTA1 protein

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block with 5% non-fat dry milk in TBST

• Primary antibody: Dilute according to manufacturer recommendations (e.g., 1:1000 to 1:16000)

• Incubation: Overnight at 4°C

• Detection: Use chemiluminescence with approximately 30 seconds exposure time

Expected results: A distinct band at approximately 34 kDa representing VTA1 . Validated cell lines for Western blot include HEK-293, A549, HepG2, K-562, SMMC-7721, and human brain tissue samples .

How can I optimize immunoprecipitation experiments using VTA1 antibody?

For successful immunoprecipitation of VTA1 and associated proteins:

• Lysate preparation: Use 0.5-1.0 mg of whole cell lysate prepared with NETN lysis buffer

• Antibody amount: Add 0.5-4.0 μg of VTA1 antibody per IP reaction

• Incubation: Overnight at 4°C with gentle rotation

• Bead addition: Add protein A/G beads and incubate for 2-4 hours

• Washing: Perform multiple washes to reduce background

• Loading: Load approximately 20% of IP for Western blot detection

This approach is particularly valuable for studying VTA1's interactions with VPS4A/B, CHMP4C, and ANCHR in the abscission checkpoint complex . HEK-293 cells have been validated for successful VTA1 immunoprecipitation .

What immunofluorescence protocol works best for visualizing VTA1 localization?

For optimal immunofluorescence visualization of VTA1:

• Cell preparation: Culture cells on coverslips to 50-70% confluence

• Fixation: Fix with 4% paraformaldehyde for 15 minutes

• Permeabilization: Use 0.1% Triton X-100 in PBS for 5 minutes

• Blocking: Block with 5% normal serum for 1 hour

• Primary antibody: Dilute VTA1 antibody 1:200-1:800

• Secondary antibody: Use fluorophore-conjugated secondary antibody

• Counterstaining: DAPI for nuclear visualization

• Analysis: Confocal microscopy for detailed subcellular localization

HeLa cells have been validated for immunofluorescence applications with VTA1 antibodies . Expected localization includes cytoplasmic distribution with potential enrichment at endosomal structures and the intercellular bridge during cytokinesis .

What are common issues with VTA1 antibodies and how can they be resolved?

When working with VTA1 antibodies, researchers may encounter these common issues:

When troubleshooting, consider including appropriate positive controls (e.g., HEK-293 cells) which consistently express detectable levels of VTA1 .

How do I interpret VTA1 localization patterns in different experimental conditions?

VTA1 localization patterns can provide valuable insights into its function:

• Normal patterns:

  • Cytoplasmic distribution with punctate structures (endosomes)

  • Recruitment to the intercellular bridge during cytokinesis

  • Co-localization with VPS4A/B at membrane scission sites

• Context-dependent patterns:

  • Extended localization at the intercellular bridge in cells with lagging chromatin

  • Co-localization with CHMP4C and ANCHR in the abscission checkpoint complex

  • Potential recruitment to virus budding sites during viral infection

• Abnormal patterns:

  • Absence from the intercellular bridge may indicate cytokinesis defects

  • Altered co-localization with VPS4 might reflect disrupted protein-protein interactions

Research shows that VTA1 knockout affects abscission timing and increases the percentage of intercellular bridges containing lagging chromatin, indicating its role in the abscission checkpoint .

How can I verify VTA1 antibody specificity?

Validating VTA1 antibody specificity is crucial for reliable results:

• Genetic validation approaches:

  • VTA1 knockout cells as negative controls

  • siRNA knockdown to reduce VTA1 expression

  • Rescue experiments with exogenous VTA1 expression

• Biochemical validation:

  • Immunoprecipitation followed by mass spectrometry

  • Testing multiple antibodies targeting different epitopes

  • Pre-adsorption with recombinant VTA1 protein

• Expected observations:

  • Specific 34 kDa band in Western blot

  • Characteristic subcellular localization in immunofluorescence

  • Reproducible protein interactions in immunoprecipitation

Proper validation ensures that phenotypes observed are due to specific detection of VTA1 rather than antibody cross-reactivity.

How can VTA1 antibodies be used to study the abscission checkpoint?

VTA1 antibodies offer powerful tools for investigating the abscission checkpoint:

• Co-immunoprecipitation experiments:

  • VTA1 interacts with checkpoint proteins ANCHR and CHMP4C

  • VTA1 knockout disrupts the interaction between VPS4A and ANCHR

  • VPS4A knockout disrupts VTA1's ability to interact with CHMP4C

• Immunofluorescence applications:

  • Monitor VTA1 recruitment to the intercellular bridge

  • Measure abscission timing in wild-type versus VTA1 knockout cells

  • VTA1 knockout cells show accelerated abscission

  • VTA1 knockout increases intercellular bridges with lagging chromatin

• Structure-function analysis:

  • Compare wild-type versus mutant VTA1 in restoring normal abscission timing

  • Identify domains critical for checkpoint function

Research has demonstrated that VTA1 is an integral part of the CHMP4C/ANCHR/VPS4A abscission checkpoint complex involved in regulating abscission timing .

What approaches can be used to study VTA1's interactions with ESCRT-III proteins?

To investigate VTA1's interactions with ESCRT-III components:

• Structural approaches:

  • The N-terminal domain of VTA1 contains two MIT motifs that interact with ESCRT-III proteins

  • Key residues Trp-122 and Lys-152 are important for VTA1 interaction with Did2 and Vps60

  • Surface grooves formed by helix 2 and helix 3 of the MIT motifs mediate protein-protein interactions

• Mutagenesis studies:

  • Create mutations in conserved surface residues (e.g., Trp-122, Lys-152)

  • Assess binding to GST-Did2 and GST-Vps60 using pull-down assays

  • Mutations in these key residues reduce binding to both Did2 and Vps60

• Functional assays:

  • Examine MVB sorting defects in cells expressing mutant VTA1

  • Assess effects on ESCRT-III assembly and disassembly dynamics

Understanding these interactions helps elucidate how VTA1 coordinates ESCRT-III assembly and disassembly during membrane remodeling events.

How does VTA1 regulate VPS4 activity and what methods can be used to study this?

VTA1's regulation of VPS4 can be studied through these approaches:

• Biochemical characterization:

  • VTA1 binds to VPS4A and VPS4B, with stronger binding affinity for VPS4A

  • The C-terminal domain of VTA1 promotes ATP-dependent double ring assembly of Vps4

  • VTA1 dimerization is necessary for its function as a Vps4 regulator

• Structural analysis:

  • The C-terminal domain mediates VTA1 dimerization

  • Both subunits in the VTA1 dimer are necessary for function

  • Mutagenesis of key interface residues can disrupt dimerization

• Functional studies:

  • Native PAGE reveals lower molecular weight bands for VPS4 in VTA1 knockout cells, indicating decreased levels of hexameric VPS4

  • VTA1 knockout does not affect cellular levels or localization of VPS4A and VPS4B

These approaches provide mechanistic insights into how VTA1 promotes VPS4 assembly and activity during membrane remodeling events.

What is the role of VTA1 in HIV-1 budding and how can it be studied?

VTA1's role in HIV-1 budding can be investigated through:

• Cellular assays:

  • Measure virus release efficiency in cells with normal versus depleted VTA1

  • Analyze viral particle morphology by electron microscopy

  • Track VTA1 recruitment to HIV-1 budding sites

• Biochemical approaches:

  • Co-immunoprecipitation of VTA1 with viral Gag protein

  • Assess interaction dependencies with other ESCRT components

  • Identify specific domains of VTA1 required for virus budding

• Structural implications:

  • The mechanism of VTA1 regulation appears conserved from yeast to humans

  • VTA1 interactions with VPS4 and ESCRT-III proteins are important for retrovirus release

VTA1 is known to be involved in HIV-1 budding , likely through its role in coordinating ESCRT-III and VPS4 activities during membrane scission events.

How do the functions of VPS4A and VPS4B isoforms differ in relation to VTA1?

Recent research has revealed important distinctions between VPS4 isoforms:

• Functional differences:

  • VPS4A has unique regulatory functions in cytokinesis that VPS4B cannot compensate for

  • This explains neurodevelopmental defects observed in VPS4A deficiency

• VTA1 interaction patterns:

  • VTA1 binds both VPS4A and VPS4B

  • Stronger binding affinity is observed for VPS4A compared to VPS4B

  • VTA1 also associates with monomeric VPS4A

• Cytokinesis roles:

  • VPS4A-specific interactions with ANCHR are dependent on VTA1

  • VPS4B cannot substitute for VPS4A in this checkpoint complex

These findings highlight the specialized functions of VPS4 isoforms and their differential reliance on VTA1 as a cofactor.

What methodological approaches are most effective for studying VTA1 in the context of cell division?

To study VTA1's role in cell division:

• Live-cell imaging methods:

  • Track fluorescently-tagged VTA1 during cytokinesis

  • Measure abscission timing in various experimental conditions

  • Monitor recruitment dynamics to the intercellular bridge

• Genetic approaches:

  • Compare wild-type, VTA1 knockout, and rescue cell lines

  • VTA1 knockout leads to accelerated abscission and increased frequency of intercellular bridges with lagging chromatin

  • Exogenously expressed VTA1 restores normal abscission timing

• Biochemical techniques:

  • Immunoprecipitation to study cytokinesis-specific protein complexes

  • Phosphorylation analysis during different cell cycle stages

  • Chromosome segregation analysis in VTA1-depleted cells

These approaches have revealed VTA1's unexpected role in slowing abscission as part of the abscission checkpoint mechanism .

How can contradictory data about VTA1 function be reconciled in experimental design?

When addressing contradictory findings about VTA1:

• Context-dependent functions:

  • VTA1 plays different roles in endosomal sorting versus cytokinesis

  • Cell type-specific effects may occur due to varying expression of interacting partners

  • The unexpected finding that VTA1 knockout accelerates abscission rather than delaying it highlights the complexity of its function

• Experimental design considerations:

  • Use multiple cell lines to establish consistent phenotypes

  • Employ both loss-of-function and rescue approaches

  • Validate key findings with different antibodies and methodologies

• Reconciliation approaches:

  • Design experiments that simultaneously assess multiple VTA1 functions

  • Consider post-translational modifications that might regulate VTA1 activity

  • Examine protein complex composition in different cellular contexts

Understanding these context-dependent functions is essential for interpreting seemingly contradictory experimental results.