WIPI1 Antibody

What is WIPI1 and what is its specific role in the autophagy machinery?

WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) is a component of the autophagy machinery that controls the major intracellular degradation process by which cytoplasmic materials are packaged into autophagosomes and delivered to lysosomes for degradation . It functions downstream of the ULK1 and PI3-kinases that produce phosphatidylinositol 3-phosphate (PtdIns3P) on membranes of the endoplasmic reticulum once activated .

WIPI1 specifically:

• Binds phosphatidylinositol 3-phosphate (PtdIns3P) and potentially other phosphoinositides including PtdIns3,5P2 and PtdIns5P

• Is recruited to phagophore assembly sites at endoplasmic reticulum membranes

• Assists WIPI2 in recruiting the ATG12-ATG5-ATG16L1 complex, which directly controls the elongation of the nascent autophagosomal membrane

• Together with WDR45/WIPI4, promotes ATG2 (ATG2A or ATG2B)-mediated lipid transfer by enhancing ATG2-association with PI3P-containing membranes

Unlike WIPI2, cells lacking WIPI1 can still undergo autophagosome formation since WIPI2 is sufficient to enable recruitment of the ATG16L1 complex to the phagophore . WIPI1 appears to serve more as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity .

What are the most reliable detection methods for WIPI1 in experimental systems?

Western Blotting:

• Antibody dilution: 1:500-1:2000 is typically recommended

• Expected molecular weight: 48-49 kDa

• Validated cell lysates: A375, BxPC-3, 293T, C6, RAW 264.7, NIH/3T3, JAR, SW 1990, and HEK-293 cells

Immunohistochemistry:

• Recommended dilution: 1:50-1:500

• Antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

• Validated tissues: Human colon tissue, intrahepatic cholangiocarcinoma tissue, and ovarian cancer tissue

Immunofluorescence:

• GFP-WIPI1 expression systems can be used to visualize WIPI1 puncta as markers of autophagosome formation

• Counting WIPI1 puncta provides a quantitative method for assessing autophagy induction

mRNA Detection:

• qRT-PCR for WIPI1 mRNA levels can serve as an indicator of autophagosome formation

• WIPI1 mRNA elevation follows a time-dependent pattern similar to autophagosome accumulation

How can I differentiate between WIPI1 and other WIPI family proteins in my experiments?

Molecular Characteristics for Differentiation:

For experimental differentiation:

• Use isoform-specific antibodies that target unique regions

• Perform siRNA-mediated knockdown validation experiments with isoform-specific primers

• In functional experiments, WIPI1 knockdown shows ~50% reduction in transferrin recycling, while WIPI2, WIPI3, and WIPI4 knockdowns show only minor effects (80-90% recycling compared to 92% in control cells)

• WIPI1 knockout increases both number and size of EEA1-positive early endosomes and LAMP1-positive late endosomes/lysosomes, which is a distinct phenotype

How does WIPI1 contribute to endosomal trafficking beyond its role in autophagy?

WIPI1 has distinct roles in endosomal trafficking that appear to be interconnected with yet separate from its autophagy functions:

• Formation and Fission of Tubulo-vesicular Endosomal Transport Carriers:

  • WIPI1 specifically promotes protein exit from early endosomes toward lysosomes, the Golgi, and the plasma membrane

  • siRNA-mediated knockdown of WIPI1 leads to increased number and size of EEA1-positive early endosomal compartments (30-40% increase) and LAMP1-positive late endosomes/lysosomes (30% increase)

  • This phenotype can be rescued by expressing siRNA-resistant WIPI1 but not by WIPI1[FAAG], a version with substituted arginines critical for lipid binding

• Recycling Pathway Regulation:

  • WIPI1 knockout cells show reduced transferrin receptor (TFRC) at the cell surface

  • While endocytosis rates remain normal, WIPI1 knockout significantly impairs recycling of transferrin from endosomes back to the plasma membrane (50% reduction compared to control)

  • This function appears largely independent of other WIPI isoforms and their interactors such as ATG2 and ATG16L1

• Retrograde Transport:

  • WIPI1 knockout cells exhibit strong delays in the arrival of Shiga toxin to the Golgi complex

  • After 30 minutes of chase, 50% of Shiga toxin remains outside the Golgi in WIPI1 knockout cells compared to almost complete Golgi colocalization in control cells

• Regulation of Protein Trafficking:

  • WIPI1 may regulate protein trafficking in the mannose-6-phosphate receptor (MPR) recycling pathway

  • It localizes to cytoplasmic vesicles, endosomes, clathrin-coated vesicles, and the trans-Golgi network

These functions highlight WIPI1's multifaceted role in cellular trafficking processes beyond canonical autophagy.

What is the role of WIPI1 in the ABL-ERK-MYC signaling axis and how can it be experimentally investigated?

The ABL-ERK-MYC Signaling Pathway Regulating WIPI1:

The ABL-ERK-MYC signaling axis negatively regulates WIPI1 expression and thereby controls autophagy levels:

• ABL1 acts as a novel inhibitor of WIPI1 puncta formation

• An ABL-ERK2-MYC pathway represses WIPI1 gene expression

• ERK-controlled MYC binds to the WIPI1 promoter and represses WIPI1 mRNA synthesis

• When this signaling is counteracted, increased WIPI1 gene expression enhances autophagy

Experimental Investigation Approaches:

• Pharmacological Modulation:

  • Use dasatinib or imatinib (ABL inhibitors) to increase WIPI1 expression

  • Use DPH (small-molecule allosteric activator of ABL) to decrease WIPI1 expression

  • Validate ABL activity by monitoring phosphorylation of CRKL, a bona fide ABL substrate

• Genetic Manipulation:

  • Perform siRNA-mediated knockdown of ABL1/2 (3-fold increase in WIPI1 mRNA)

  • Target ERK and MYC with siRNA to observe effects on WIPI1 expression

  • Use WIPI1 promoter reporter constructs to directly measure MYC-mediated repression

• Visualization Techniques:

  • Monitor GFP-WIPI1 puncta formation under different treatments

  • Quantify WIPI1, WIPI2, LC3/GABARAP, and p62 as autophagy markers

• Gene Expression Analysis:

  • Use human autophagy pathway-focused gene expression profiling to assess WIPI1 regulation

  • Perform qRT-PCR to validate changes in WIPI1 mRNA levels

  • Examine effects on other autophagy genes to determine specificity

• Model Organism Validation:

  • Use C. elegans to study ABL deficiency effects on the WIPI1 ortholog ATG-18

  • Assess lifespan extension and dependency on ATG-18 expression

How can WIPI1 mRNA levels be used as an indicator of autophagosome formation?

WIPI1 mRNA levels provide a convenient and reliable method for detecting autophagosome formation, particularly in the following research scenarios:

Methodological Approach:

• Extract total RNA from cells treated with autophagy inducers

• Perform quantitative real-time RT-PCR for WIPI1 and MAP1LC3B mRNA

• Compare time-dependent changes in mRNA levels with autophagosome formation

Key Experimental Findings:

• WIPI1 mRNA is induced prior to the accumulation of the autophagy marker protein MAP1LC3 in thapsigargin- and C2-ceramide-treated cells

• Time-dependent WIPI1 mRNA elevation closely follows autophagosome accumulation patterns

• Transcriptional attenuation of WIPI1 mRNA using RNA interference inhibits autophagosome formation (measured by puncta counting) in thapsigargin-treated cells

• WIPI1 mRNA increase is consistently observed across various cell types, including human fibroblasts (WI-38 and TIG-1), human cancer cells (U-2 OS, Saos-2, and MCF7), and rodent fibroblasts (Rat-1)

Validation with Autophagy Inhibitors:

• Saturating concentrations of bafilomycin A1 (25 nM) or chloroquine (50 μM) result in accumulation of WIPI1 mRNA, but to a lesser extent than with thapsigargin treatment

• Unsaturating concentrations of these inhibitors (2.5 nM bafilomycin A1 or 10 μM chloroquine) do not significantly affect WIPI1 mRNA levels

Time-Course Analysis:

• In A549 cells treated with 100 μM C2-ceramide or 0.5 μM thapsigargin, WIPI1 and MAP1LC3B mRNA levels peak after 12 hours of treatment and decrease by 24 hours

• Early changes in WIPI1 mRNA can be detected as soon as 1-4 hours after treatment

This approach offers advantages over protein-based autophagy detection methods, particularly for high-throughput screening applications.

What techniques can be used to investigate the functional relationship between WIPI1 and WIPI2 in autophagosome formation?

Experimental Approaches to Study WIPI1-WIPI2 Functional Relationship:

• Genetic Knockout/Knockdown Studies:

  • Generate WIPI-knockout (single, double, and quadruple knockout) cell lines using CRISPR-Cas9

  • Compare phenotypes between different knockout combinations

  • Use siRNA-mediated knockdown to achieve temporary depletion for specific experiments

  • Perform rescue experiments with different WIPI isoforms to determine functional redundancy

• Protein-Protein Interaction Analysis:

  • Investigate WIPI1-WIPI2 heterodimerization using co-immunoprecipitation

  • Perform proximity ligation assays to detect interactions in situ

  • Use FRET or BRET techniques to measure dynamic interactions

  • Study interactions with ATG16L1 complex to understand differential recruitment

• Structure-Function Analysis:

  • Express WIPI1[FAAG] mutants with altered lipid-binding capacity and assess effects on function

  • Compare the different isoforms: WIPI1A versus WIPI2B and WIPI2D recruitment to phagophores

  • Use domain swapping between WIPI1 and WIPI2 to identify regions responsible for specific functions

• Lipid Binding Assays:

  • Compare binding affinities of WIPI1 and WIPI2 to phosphoinositides using protein-lipid overlay assays

  • Use liposome flotation assays to quantify membrane association

  • Perform surface plasmon resonance to measure binding kinetics

• Advanced Imaging Techniques:

  • Use correlative light electron microscopy (CLEM) to visualize WIPI1 and WIPI2 localization on forming autophagosomes

  • Perform live-cell imaging to track the sequential recruitment of WIPI1 and WIPI2

  • Apply super-resolution microscopy to resolve spatial organization on the phagophore

• Functional Readouts:

  • Measure LC3 lipidation levels as a downstream effect of WIPI1/2 function

  • Assess autophagic flux using tandem-fluorescent LC3 or Halo-GFP processing assays

  • Quantify the formation of WIPI1/2 puncta under various autophagy-inducing conditions

• Innovative Applications:

  • Study the transport of WIPI1-positive autophagic membranes through tunneling nanotubes to cells with deficient autophagy

  • Investigate how WIPI1 enhances phagophore formation in coordination with WIPI2

  • Examine regulatory mechanisms specific to WIPI1 versus those affecting both WIPI proteins

What are common issues in WIPI1 antibody experiments and how can they be resolved?

Common Issues and Solutions:

Best Practices for Validation:

• Antibody Validation:

  • Perform knockdown/knockout controls to confirm specificity

  • Test antibodies in multiple applications (WB, IHC, IF)

  • Compare results using antibodies targeting different epitopes

  • Include non-specific IgG controls

• Expression Controls:

  • Use recombinant WIPI1 as a positive control in Western blots

  • Include cells with known WIPI1 expression levels (A375, BxPC-3, 293T)

  • Compare results across multiple cell lines

  • Test both starved and non-starved conditions

• Application-Specific Recommendations:

  • For Western blotting: Use RIPA buffer with phosphatase inhibitors

  • For IHC: Optimize antigen retrieval (test both pH 6.0 and pH 9.0 buffers)

  • For IF: Include co-staining with established autophagy markers

  • For mRNA analysis: Design primers spanning exon-exon junctions

How do experimental conditions affect WIPI1 detection and function?

Understanding how various experimental conditions influence WIPI1 detection and function is crucial for designing robust experiments:

Autophagy Induction Conditions:

Cell Type Variations:

WIPI1 expression and function varies significantly across cell types:

• Human fibroblasts (WI-38, TIG-1): Reliable WIPI1 induction with thapsigargin/C2-ceramide

• Human cancer cells (U-2 OS, Saos-2, MCF7): High baseline expression in some cancer lines

• Rodent cells (Rat-1, NIH/3T3): Show conserved WIPI1 responses across species

• HEK293T cells: Frequently used for WIPI knockout studies

• A375, BxPC-3, 293T: Recommended for Western blot detection of endogenous WIPI1

Technical Factors Affecting Detection:

• Antibody selection:

  • Monoclonal antibodies (e.g., [EPR8110]) provide higher specificity

  • Polyclonal antibodies may detect multiple isoforms

  • Most antibodies target C-terminal regions of WIPI1

• Fixation methods:

  • For IF: 4% paraformaldehyde preserves WIPI1 puncta structure

  • For IHC: Paraffin embedding requires effective antigen retrieval

• Lysis conditions:

  • Phosphatase inhibitors should be included to preserve phosphorylation status

  • Gentle lysis methods help maintain protein-protein interactions

  • Membrane fractionation may enhance detection of membrane-bound WIPI1

• Expression systems:

  • GFP-WIPI1 tends to homodimerize with endogenous WIPI1

  • Overexpression may alter normal distribution and function

  • WIPI1A isoform is most commonly used in research

What criteria should be used to validate WIPI1 involvement in specific autophagy pathways?

To rigorously validate WIPI1's role in specific autophagy pathways, researchers should employ multiple complementary approaches:

1. Genetic Validation:

• Generate WIPI1 knockout cells using CRISPR-Cas9 technology

• Create knockdown models using siRNA or shRNA with appropriate controls

• Perform rescue experiments with wild-type and mutant WIPI1 constructs

• Utilize WIPI1[FAAG] mutant (with disrupted lipid-binding sites) as a negative control

2. Functional Autophagy Assessments:

3. Molecular Interaction Evidence:

• Demonstrate WIPI1 binding to phosphoinositides (PtdIns3P, PtdIns5P)

• Confirm interactions with the autophagy machinery (WIPI2, ATG2, ATG16L1)

• Map the timing of WIPI1 recruitment relative to other autophagy proteins

• Study the dependency on upstream regulators (ULK1, VPS34, ABL)

4. Pathway-Specific Criteria:

For starvation-induced autophagy:

• WIPI1 puncta formation should increase upon nutrient deprivation

• Effect should be blocked by PI3K inhibitors (wortmannin, 3-MA)

• WIPI1 should colocalize with DFCP1-positive omegasomes

For mitophagy:

• WIPI1 should colocalize with damaged mitochondria

• Recruitment should depend on PINK1/Parkin for mitochondrial damage

• Effects should be distinct from general autophagy responses

For xenophagy:

• WIPI1 should localize to bacteria-containing vesicles

• WIPI1 knockdown should affect bacterial clearance

• Response should be specific to certain bacterial species (e.g., S. aureus)

5. Signaling Pathway Integration:

• Verify WIPI1 regulation by the ABL-ERK-MYC axis through both genetic and pharmacological approaches

• Assess WIPI1 response to AMPK and TORC1 modulation

• Determine whether WIPI1 functions downstream of calcium signaling in thapsigargin-induced autophagy

6. Evolutionary Conservation:

• Compare WIPI1 function with its ortholog ATG-18 in C. elegans

• Evaluate lifespan effects of WIPI1/ATG-18 modulation across species

• Assess conservation of regulatory mechanisms between human WIPI1 and yeast/nematode orthologs

How does WIPI1 contribute to intercellular communication through tunneling nanotubes?

Recent research has revealed an unexpected role for WIPI1 in intercellular communication through tunneling nanotubes (TNTs), providing new insights into how autophagic activity might be coordinated between cells:

Key Findings:

• WIPI1-positive autophagic membranes can be transported through tunneling nanotubes (TNTs) to neighboring cells with low autophagic activity

• This transport appears to be enhanced when WIPI1 expression is increased due to counteracting the ABL-ERK-MYC signaling axis

• Autophagic membranes positive for WIPI1, WIPI2, or LC3 have been observed within TNTs

• In coculture experiments, these membranes can be transported to cells lacking sufficient autophagy, such as ATG16L-deficient human cells

Experimental Approaches to Study This Phenomenon:

• Coculture Systems:

  • Setup cocultures with fluorescently labeled autophagy-competent cells and autophagy-deficient cells

  • Use differentially labeled WIPI1 and other autophagy markers to track intercellular transport

  • Analyze the directionality of transport (preferential movement toward autophagy-deficient cells)

• Live Imaging Techniques:

  • Perform time-lapse microscopy to visualize WIPI1-positive membrane movement through TNTs

  • Use photoactivatable or photoconvertible fluorescent WIPI1 constructs to track specific pools

  • Apply super-resolution microscopy to characterize the structure of TNTs containing autophagic membranes

• Functional Assessment:

  • Measure the restoration of autophagic activity in deficient cells after coculture

  • Determine whether transported membranes contribute to functional autophagosomes

  • Assess whether this mechanism can protect autophagy-deficient cells from stress

• Mechanistic Studies:

  • Investigate the molecular machinery required for TNT formation in the context of WIPI1

  • Determine whether WIPI1 plays an active role in directing membrane transport or is merely a cargo

  • Identify proteins that regulate the selectivity and efficiency of this intercellular communication

• Physiological Relevance:

  • Examine this phenomenon in tissue contexts where cell-cell communication is crucial

  • Investigate whether dysfunctional intercellular autophagy transport contributes to disease

  • Study how aging or stress conditions affect WIPI1-mediated intercellular communication

What is the relevance of WIPI1 in lifespan regulation and age-related pathologies?

The discovery that WIPI1 and its orthologs play roles in lifespan regulation opens exciting new research directions:

Evidence for WIPI1's Role in Lifespan Regulation:

• ABL deficiency in C. elegans increases gene expression of the WIPI1 ortholog ATG-18

• This increase in ATG-18 expression correlates with prolonged lifespan in the nematodes

• The lifespan extension is dependent on ATG-18, suggesting a causal relationship

• WIPI1 appears to act as an enhancer of autophagy that is physiologically relevant for regulating autophagic activity over the lifespan

Research Approaches to Investigate This Connection:

• Comparative Studies Across Model Organisms:

  • Extend findings from C. elegans to Drosophila, zebrafish, and mammalian models

  • Determine whether WIPI1 upregulation consistently correlates with extended lifespan

  • Compare the effects of WIPI1 modulation with established lifespan extension interventions (caloric restriction, rapamycin)

• Tissue-Specific Analysis:

  • Investigate tissue-specific roles of WIPI1 in aging (brain, muscle, liver, etc.)

  • Determine if WIPI1 expression changes with age in different tissues

  • Create tissue-specific WIPI1 overexpression or knockout models to assess localized effects

• Connection to Age-Related Pathologies:

  • Study WIPI1's role in neurodegenerative diseases where autophagy is implicated

  • Investigate WIPI1 in metabolic disorders and cardiovascular diseases

  • Examine WIPI1 expression in senescent cells and its potential role in senescence-associated secretory phenotype

• Molecular Mechanisms:

  • Determine how WIPI1-enhanced autophagy specifically contributes to longevity

  • Investigate whether WIPI1 affects selective autophagy of age-damaged organelles

  • Study interactions between WIPI1 and known longevity pathways (insulin/IGF-1, AMPK, sirtuins)

• Translational Applications:

  • Develop small molecules that enhance WIPI1 expression or activity

  • Test whether pharmacological inhibition of the ABL-MYC axis to increase WIPI1 levels affects lifespan in mammals

  • Investigate WIPI1 as a biomarker for biological aging or autophagy capacity

• Potential Disease Relevance:

  • WIPI1 is upregulated in various tumors, suggesting a role in carcinogenesis

  • This contrasts with its positive role in lifespan regulation, warranting investigation into context-dependent functions

  • Study how WIPI1's dual roles in normal aging and disease might be regulated

How might WIPI1 be targeted therapeutically in autophagy-related diseases?

Based on our understanding of WIPI1's functions and regulation, several therapeutic strategies could be developed for autophagy-related diseases:

Potential Therapeutic Approaches:

• Enhancing WIPI1 Expression:

  • Targeting the ABL-ERK-MYC axis with existing drugs:

    • ABL inhibitors (dasatinib, imatinib) increase WIPI1 expression and autophagy

    • MEK/ERK inhibitors might increase WIPI1 by reducing MYC-mediated repression

    • MYC inhibitors could directly relieve repression of the WIPI1 promoter

  • Epigenetic modulators to counteract promoter silencing

  • RNA-based therapies to increase WIPI1 mRNA stability or translation

• Modulating WIPI1 Function:

  • Small molecules that enhance WIPI1 binding to phosphoinositides

  • Peptide-based approaches to promote WIPI1-WIPI2 interactions

  • Compounds that stabilize WIPI1 protein or prevent its degradation

• Disease-Specific Applications:

• Considerations for Therapeutic Development:

  • Cell/tissue specificity of delivery to avoid unintended effects

  • Optimal timing of intervention during disease progression

  • Combination with other autophagy modulators or standard therapies

  • Biomarkers to identify patients likely to respond (e.g., low baseline WIPI1 levels)

• Innovative Delivery Approaches:

  • Exploit WIPI1's role in tunneling nanotubes to deliver therapeutic cargo between cells

  • Develop nanoparticles that mimic WIPI1's membrane-binding properties

  • Consider cell-based therapies with enhanced WIPI1 expression

• Potential Challenges:

  • Achieving appropriate levels of autophagy enhancement without cytotoxicity

  • Managing context-dependent roles of WIPI1 in different tissues

  • Addressing compensatory mechanisms by other WIPI family members

  • Developing specific WIPI1 modulators without off-target effects