ATG18C Antibody

What is ATG18/WIPI-2 and what is its primary function in cellular processes?

ATG18, also known as WIPI-2, is a key protein involved in autophagy that regulates the assembly of multiprotein complexes through its characteristic 7-bladed propeller structure, which facilitates interactions with phospholipids. It functions as a mammalian effector of phosphatidylinositol 3-phosphate (PtdIns3P) and is ubiquitously expressed across various cell lines. ATG18/WIPI-2 plays a critical role in early autophagosomal formation by being recruited to initial structural components alongside Atg16 and ULK1, and is essential for the formation of LC3-positive autophagosomes .

The protein is approximately 49 kDa in size, though it's important to note that six isoforms exist with varying molecular weights. In the broader context of cellular regulation, ATG18/WIPI-2 participates in both selective and non-selective autophagy pathways, making it a crucial target for researchers investigating autophagy-related processes .

How does ATG18/WIPI-2 interact with phospholipids and why is this interaction significant?

ATG18/WIPI-2 interacts with phospholipids, particularly phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂), through its β-barrel structure. Unlike proteins with distinct phosphoinositide-binding modules, ATG18 requires an intact β-barrel comprising almost the entire length of the protein to associate with these lipids. The FRRG motif located on the β-barrel is a strong candidate for the direct site of interaction with PtdIns(3,5)P₂, as mutation of the double arginines disrupts binding and shifts ATG18 localization from the vacuole to the cytosol .

This interaction is significant because:

• It allows ATG18 to function as a "sensor" for specific membrane lipids

• It mediates the protein's recruitment to autophagic membranes

• It regulates ATG18's role in membrane remodeling during autophagy

• It enables feedback regulation of Fab1 kinase activity, affecting PtdIns(3,5)P₂ levels

Research using an ATG18 variant (ATG18(FTTG)) that cannot bind phosphoinositides has demonstrated that PtdIns(3)P binding is essential for full activity in both selective and nonselective autophagy, highlighting the critical nature of this interaction for proper autophagic function .

What are the technical specifications of ATG18/WIPI-2 antibodies used in research applications?

The Anti-ATG18 (WIPI-2) antibody (clone 2A2) commonly used in research has the following technical specifications:

For immunohistochemistry applications, this antibody has been validated to detect ATG18 in human colorectal adenocarcinoma tissue. In Western blot applications, it has been shown to detect ATG18 in human testis tissue lysate at a concentration of 10 μg .

How should I design experiments to study ATG18/WIPI-2 interactions with binding partners?

When designing experiments to study ATG18/WIPI-2 interactions with binding partners, consider implementing the following methodological approaches based on established research protocols:

Co-immunoprecipitation Protocol:

• Grow cells to mid-logarithmic phase (OD₆₀₀ ~0.6) and treat with rapamycin for 10 minutes if studying autophagy-induced interactions

• Convert cells to spheroplasts while maintaining them in rapamycin-containing buffers

• Lyse spheroplasts by osmotic shock in immunoprecipitation buffer (50 mM Tris-HCl (pH 8.0), 200 mM sorbitol, 150 mM KCl, 5 mM MgCl₂, 0.5 mg/ml BSA, 1% Triton X-100, plus protease and phosphatase inhibitors)

• Remove cell debris by centrifugation at 500 × g for 5 minutes

• Centrifuge samples at 100,000 × g for 30 minutes

• Incubate supernatants with appropriate antibodies (e.g., anti-Atg2 or anti-HA for tagged ATG18)

• Analyze precipitated complexes by SDS-PAGE followed by immunoblotting with antibodies against suspected binding partners

Cross-linking Immunoprecipitation:

• Metabolically label cells with ³⁵S

• Prepare osmotic whole cell lysates

• Treat lysates with cross-linker DSP [dithiobis(succinimidyl-propionate)]

• TCA-precipitate proteins

• Perform two successive overnight immunoprecipitations using anti-HA antibody (if using HA-tagged ATG18)

• Cleave the cross-linker and resolve samples by SDS-PAGE

• Use autoradiography to reveal cross-linked proteins

Using these approaches, researchers have successfully identified interactions between ATG18 and proteins such as Atg2 and Vac17. When designing your own experiments, consider including appropriate controls such as wild-type vs. mutant forms of ATG18 (e.g., the FTTG variant that cannot bind phosphoinositides) to assess the functional significance of identified interactions .

What methods are most effective for visualizing ATG18/WIPI-2 localization during autophagy?

For effective visualization of ATG18/WIPI-2 localization during autophagy, researchers should consider the following methodological approaches:

Fluorescent Microscopy with Tagged Constructs:

• Generate fluorescent protein-tagged ATG18 constructs (e.g., ATG18-GFP, ATG18-HA-GFP)

• Express constructs in appropriate cell models

• Induce autophagy using standard methods (e.g., rapamycin treatment, starvation)

• Visualize using fluorescence microscopy with appropriate filter sets (e.g., for GFP: FF495-Di02 dichroic mirror, FF01-472/30 excitation filter)

• For multi-color imaging with markers like mRFP, use compatible filter sets (e.g., FF593-Di02 dichroic mirror, FF593-Em02 excitation filter)

Domain-specific Localization Studies:
To investigate the role of specific domains in ATG18 localization, researchers can use:

• Site-directed mutagenesis to create variants (e.g., ATG18(FTTG) that cannot bind phosphoinositides)

• Domain fusion constructs (e.g., ATG18-FYVE domain fusions) to investigate membrane targeting requirements

• Co-localization with known autophagosomal markers (e.g., LC3, Atg2, Atg16)

Biochemical Fractionation:

• Separate cellular components through differential centrifugation

• Isolate membrane fractions associated with autophagosome formation

• Detect ATG18/WIPI-2 in fractions using western blotting

These approaches enable researchers to precisely track the spatial and temporal dynamics of ATG18/WIPI-2 during autophagy induction and progression. Additionally, combining these methods with functional assays can provide insights into how localization correlates with autophagic activity .

How can I quantitatively assess ATG18/WIPI-2 activity in autophagy assays?

Quantitative assessment of ATG18/WIPI-2 activity in autophagy assays requires multiple complementary approaches to capture both direct protein function and its downstream effects on autophagic processes:

Biochemical Approaches:

• Phosphoinositide Binding Assays: Measure binding affinity of purified ATG18/WIPI-2 to PtdIns3P and PtdIns(3,5)P₂ using protein-lipid overlay assays or liposome binding assays

• Western Blot Analysis: Quantify changes in autophagy markers like LC3-II/I ratio in wild-type vs. ATG18-depleted or mutant (e.g., FTTG variant) backgrounds

• ApeI Processing Assay: Monitor the processing of ApeI (aminopeptidase I) by immunoblotting as a measure of selective autophagy function in yeast systems

Microscopy-Based Quantification:

• Puncta Formation: Count ATG18-GFP positive puncta per cell as a measure of recruitment to autophagic structures

• Co-localization Analysis: Quantify co-localization coefficients between ATG18 and markers like Atg2 or LC3

• Autophagic Body Accumulation: In yeast studies, use phase-contrast microscopy to quantify accumulation of autophagic bodies in the vacuole as a measure of autophagy progression

Functional Readouts:

• Autophagy Flux Assays: Measure autophagic flux using tandem-tagged LC3 reporters (mRFP-GFP-LC3) in the presence/absence of ATG18/WIPI-2

• Selective Autophagy Assays: Quantify clearance of specific cargo (e.g., protein aggregates, damaged mitochondria) as a function of ATG18/WIPI-2 activity

How does ATG18/WIPI-2 function differ between selective and non-selective autophagy pathways?

ATG18/WIPI-2 participates in both selective and non-selective autophagy, but with nuanced differences in its recruitment, regulation, and functional impact:

Common Functional Aspects:

• PtdIns3P binding is essential for ATG18 activity in both pathways

• ATG18 forms a complex with Atg2 in both selective and non-selective autophagy

• Both pathways require proper ATG18 localization to autophagosomal membranes

Selective Autophagy-Specific Functions:

• In selective autophagy pathways like the cytoplasm-to-vacuole targeting (Cvt) pathway, ATG18 participates in cargo recognition and packaging

• ATG18 is required for proper processing of specific substrates like aminopeptidase I (ApeI)

• ATG18 may interact with cargo-specific adaptor proteins that are unique to selective autophagy

Non-selective Autophagy-Specific Functions:

• During starvation-induced autophagy, ATG18 is involved in bulk autophagosome formation

• The protein may help regulate the size and number of autophagosomes formed during non-selective autophagy

What are the methodological challenges in studying ATG18/WIPI-2 isoforms and their specific roles?

Studying ATG18/WIPI-2 isoforms presents several methodological challenges that researchers must address through carefully designed experimental approaches:

Challenge 1: Isoform-Specific Detection

• The presence of six ATG18 isoforms with varying molecular weights complicates specific detection

• Antibodies may cross-react between isoforms, making it difficult to study one isoform in isolation

• Solution: Develop and validate isoform-specific antibodies that target unique epitopes, or use genetic approaches to tag specific isoforms

Challenge 2: Functional Redundancy

• Overlapping functions between isoforms may mask phenotypes in single-isoform knockout/knockdown systems

• Solution: Implement combinatorial knockdown/knockout approaches and rescue experiments with isoform-specific constructs to delineate unique functions

Challenge 3: Tissue and Cell-Type Specificity

• Expression patterns of ATG18/WIPI-2 isoforms may vary across tissues and cell types

• Solution: Perform comprehensive expression profiling across tissues and cell types before selecting experimental models

Challenge 4: Post-Translational Modifications

• Different isoforms may undergo distinct post-translational modifications

• Solution: Use mass spectrometry-based proteomics to characterize modification patterns specific to each isoform

Challenge 5: Protein-Protein Interaction Networks

• Each isoform may engage in unique protein-protein interactions

• Solution: Perform isoform-specific immunoprecipitation followed by mass spectrometry to identify interaction partners

Technical Approach Table:

Addressing these challenges requires integrated approaches combining genetic, biochemical, and imaging techniques tailored to the specific research question about ATG18/WIPI-2 isoforms .

How do mutations in the phosphoinositide-binding motifs of ATG18/WIPI-2 affect its function in autophagy?

Mutations in the phosphoinositide-binding motifs of ATG18/WIPI-2, particularly the FRRG motif, have profound effects on its function in autophagy through several mechanisms:

Molecular Consequences of Binding Motif Mutations:

• Altered Membrane Recruitment: The FTTG mutation (where arginines in the FRRG motif are replaced with threonines) abolishes binding to phosphoinositides, resulting in mislocalization of ATG18. Instead of localizing to autophagosomal membranes, the mutant protein remains predominantly cytosolic .

• Preserved Protein-Protein Interactions: Interestingly, the ATG18(FTTG) variant still forms a complex with Atg2 in a normal manner. This indicates that phosphoinositide binding is not a prerequisite for ATG18-Atg2 complex formation, suggesting these are functionally separable aspects of ATG18 biology .

• Impaired Autophagosome Formation: Despite maintaining the ability to form complexes with Atg2, cells expressing ATG18(FTTG) show significant defects in autophagosome formation, indicating that membrane recruitment via phosphoinositide binding is essential for ATG18's function in autophagy beyond simply serving as a scaffold for Atg2 .

Functional Impact on Autophagy Pathways:

These findings have led to a model where ATG18/WIPI-2 has dual functions in autophagy: (1) as a scaffold that forms a complex with Atg2 and potentially other proteins, and (2) as a phosphoinositide effector that facilitates recruitment of this complex to autophagic membranes. Both functions are necessary for proper autophagy, but phosphoinositide binding specifically directs the complex to the appropriate membrane locations .

For researchers investigating ATG18/WIPI-2 function, these mutations provide valuable tools to dissect the relative contributions of protein-protein interactions versus lipid binding in various autophagic processes .

What are common technical issues when using ATG18/WIPI-2 antibodies in immunoblotting and immunohistochemistry?

Researchers working with ATG18/WIPI-2 antibodies may encounter several technical challenges that can affect experimental outcomes. Here are common issues and troubleshooting approaches:

For Western Blotting Applications:

For Immunohistochemistry Applications:

Critical Procedural Considerations:

• Sample Preparation: ATG18/WIPI-2 localization and expression are highly responsive to autophagy induction. Consider the autophagy status of your samples when interpreting results.

• Antibody Validation: Always include appropriate positive controls (human testis tissue lysate for WB, human colorectal adenocarcinoma tissue for IHC) and negative controls (isotype controls or tissues/cells lacking ATG18/WIPI-2).

• Fixation Methods: For immunohistochemistry, fixation methods can significantly affect epitope accessibility, particularly for the C-terminal epitope recognized by the 2A2 clone.

• Storage Conditions: Maintain antibody stability by storing at 2-8°C and avoiding repeated freeze-thaw cycles, as the antibody is stable for approximately 1 year under proper storage conditions .

How can I validate the specificity of ATG18/WIPI-2 antibodies in my experimental system?

Validating the specificity of ATG18/WIPI-2 antibodies is crucial for ensuring reliable research results. Here is a comprehensive approach to antibody validation:

Genetic Validation Approaches:

• Knockout/Knockdown Controls:

  • Generate ATG18/WIPI-2 knockout or knockdown cells/tissues

  • Compare antibody signal between wild-type and knockout/knockdown samples

  • Absence of signal in knockout/knockdown samples confirms specificity

• Overexpression Validation:

  • Express tagged versions of ATG18/WIPI-2 (e.g., HA-tagged or GFP-tagged)

  • Perform dual labeling with anti-ATG18 antibody and anti-tag antibody

  • Co-localization confirms antibody recognition of the correct protein

Biochemical Validation Methods:

• Western Blot Analysis:

  • Confirm band at expected molecular weight (~49 kDa for ATG18/WIPI-2)

  • Verify disappearance or reduction of band in knockout/knockdown samples

  • Check for detection of recombinant ATG18/WIPI-2 protein

• Immunoprecipitation:

  • Immunoprecipitate using the antibody and confirm identity of pulled-down protein by mass spectrometry

  • Perform reciprocal immunoprecipitation with a different antibody against ATG18/WIPI-2 or a known interaction partner (e.g., Atg2)

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide (C-terminal peptide of human ATG18)

  • Verify elimination of specific signal in Western blot or immunostaining

Application-Specific Validation:

• For Immunohistochemistry:

  • Test antibody on multiple tissue types with known ATG18/WIPI-2 expression patterns

  • Include autophagy-induced and basal conditions to verify expected localization changes

  • Compare with in situ hybridization for ATG18/WIPI-2 mRNA

• For Immunofluorescence:

  • Verify punctate pattern during autophagy induction

  • Confirm co-localization with known autophagosomal markers

  • Test response to autophagy inducers (e.g., rapamycin, starvation) and inhibitors

Validation Reporting Checklist:

By implementing these validation steps, researchers can ensure that experimental results obtained with ATG18/WIPI-2 antibodies accurately reflect the biology of this important autophagy protein .

What controls should I include when studying ATG18/WIPI-2 phosphoinositide binding and its impact on autophagy?

When investigating ATG18/WIPI-2 phosphoinositide binding and its relationship to autophagy, incorporating appropriate controls is essential for data interpretation and experimental validity:

Essential Experimental Controls:

• Protein Expression Controls:

  • Wild-type ATG18/WIPI-2: Serves as the positive control for normal phosphoinositide binding and function

  • ATG18(FTTG) Mutant: Key negative control for phosphoinositide binding; maintains protein-protein interactions but lacks lipid binding

  • Expression Level Verification: Confirm comparable expression levels between wild-type and mutant proteins to ensure phenotypic differences are not due to expression disparities

• Autophagy Status Controls:

  • Basal Conditions: Establish baseline ATG18/WIPI-2 localization and function

  • Autophagy Induction: Include rapamycin treatment or starvation conditions to activate autophagy

  • Autophagy Inhibition: Use PI3K inhibitors (e.g., wortmannin) to block PtdIns3P production and disrupt ATG18/WIPI-2 recruitment

• Membrane Binding Controls:

  • PtdIns3P-enriched Membranes: Positive control for ATG18/WIPI-2 recruitment

  • PtdIns3P-depleted Membranes: Created through PI3K inhibition or genetic manipulation of PI3K components

  • Other Phosphoinositide-containing Membranes: Test specificity of binding

Technical Control Approaches:

Biochemical Assay Controls:

For in vitro phosphoinositide binding assays:

• Include positive control proteins with known phosphoinositide binding properties

• Test multiple phosphoinositide species to determine binding specificity

• Include non-phosphoinositide lipids as negative controls

• Perform concentration-dependent binding studies to establish affinity parameters

Data Interpretation Considerations:

When analyzing results from ATG18/WIPI-2 phosphoinositide binding studies, consider:

• The temporal relationship between phosphoinositide binding and autophagy progression

• The potential for compensatory mechanisms in genetic knockout models

• The distinction between direct effects of phosphoinositide binding versus secondary effects on protein-protein interactions

• The possibility that different experimental systems (yeast vs. mammalian cells) may yield varying results due to evolutionary divergence of autophagy mechanisms

What are the emerging research areas investigating ATG18/WIPI-2 in disease models?

ATG18/WIPI-2 research is expanding beyond basic autophagy mechanisms to explore its roles in various disease contexts. Several emerging research areas are particularly noteworthy:

1. Neurodegenerative Disorders:
Researchers are investigating how ATG18/WIPI-2 function affects the clearance of protein aggregates in conditions like Alzheimer's, Parkinson's, and Huntington's diseases. The focus is on whether enhancing ATG18/WIPI-2 activity could promote autophagy-mediated clearance of disease-associated protein aggregates.

2. Cancer Biology:
ATG18/WIPI-2's role in cancer is complex and context-dependent:

• In some cancers, ATG18/WIPI-2-mediated autophagy may promote tumor cell survival under stress conditions

• In others, proper ATG18/WIPI-2 function may suppress tumor initiation by maintaining cellular homeostasis

• Research is examining how ATG18/WIPI-2 expression correlates with cancer progression and treatment responses, particularly in colorectal adenocarcinoma where ATG18/WIPI-2 expression has been detected using immunohistochemistry

3. Infectious Diseases:
The role of ATG18/WIPI-2 in xenophagy (the autophagic targeting of intracellular pathogens) is an active area of investigation. Researchers are studying how pathogens may subvert ATG18/WIPI-2 function to avoid autophagic degradation.

4. Metabolic Disorders:
ATG18/WIPI-2's involvement in lipid metabolism and its interaction with vacuolar membranes suggests potential roles in metabolic regulation. Research is exploring connections between ATG18/WIPI-2 function and conditions like obesity, diabetes, and non-alcoholic fatty liver disease.

5. Aging Research:
The relationship between ATG18/WIPI-2-mediated autophagy and cellular senescence is being investigated as a potential intervention point for age-related pathologies.

Current Methodological Approaches in Disease Research:

• Generation of tissue-specific ATG18/WIPI-2 knockout models to study organ-specific functions

• Development of small molecule modulators of ATG18/WIPI-2 activity or localization

• Implementation of high-content screening to identify disease contexts where ATG18/WIPI-2 function is particularly critical

• Integration of ATG18/WIPI-2 studies with broader phosphoinositide signaling networks in disease models

As this field continues to evolve, researchers are increasingly focusing on the translational potential of ATG18/WIPI-2 as both a biomarker and therapeutic target across diverse disease contexts.

How do the structural features of ATG18/WIPI-2 determine its functional specificity in autophagy?

The structural architecture of ATG18/WIPI-2 is central to its functional specificity in autophagy, with distinct domains mediating different aspects of its activity:

Key Structural Features and Their Functions:

Structure-Function Relationships:

Structural Basis for Differential Functions:

The multifunctional nature of ATG18/WIPI-2 arises from its ability to:

• Bind phosphoinositides through the FRRG motif

• Interact with protein partners like Atg2 through surface regions

• Potentially change conformation upon membrane binding to regulate partner interactions

Future structural biology approaches, including cryo-electron microscopy of ATG18/WIPI-2 in complex with its binding partners, will likely provide additional insights into how structural features determine functional specificity in different autophagy contexts.

What technological advances might improve the study of ATG18/WIPI-2 dynamics in living cells?

Technological innovations are continuously expanding our ability to study ATG18/WIPI-2 dynamics in living cells with greater precision and physiological relevance. Several emerging approaches show particular promise:

Advanced Imaging Technologies:

• Super-Resolution Microscopy:

  • Techniques like STORM, PALM, and STED microscopy can resolve ATG18/WIPI-2 localization below the diffraction limit

  • These approaches reveal the nanoscale organization of ATG18/WIPI-2 at autophagosomal membranes

  • Implementation: Label ATG18/WIPI-2 with photoconvertible fluorophores or use antibodies conjugated to super-resolution compatible dyes

• Lattice Light-Sheet Microscopy:

  • Enables long-term 3D imaging with minimal phototoxicity

  • Particularly valuable for tracking ATG18/WIPI-2 dynamics throughout the entire autophagy process

  • Implementation: Express fluorescently-tagged ATG18/WIPI-2 and image cells using lattice light-sheet systems

• FRET/FLIM Analysis:

  • Reports on protein-protein interactions and conformational changes in real-time

  • Can reveal when and where ATG18/WIPI-2 interacts with binding partners like Atg2

  • Implementation: Generate donor-acceptor pairs with ATG18/WIPI-2 and its binding partners

Genetically Encoded Biosensors:

• Phosphoinositide Sensors:

  • Develop biosensors that report on PtdIns3P and PtdIns(3,5)P₂ dynamics in parallel with ATG18/WIPI-2 recruitment

  • Implementation: Dual-color imaging with spectrally distinct phosphoinositide probes and ATG18/WIPI-2 markers

• Split Fluorescent Protein Systems:

  • Enable visualization of ATG18/WIPI-2 complex formation with specific partners

  • Implementation: Fuse complementary fragments of split fluorescent proteins to ATG18/WIPI-2 and interaction partners

• Optogenetic Tools:

  • Allow spatiotemporal control of ATG18/WIPI-2 recruitment or activity

  • Implementation: Develop light-activatable ATG18/WIPI-2 variants or controllable phosphoinositide production systems

Emerging Biochemical and Genetic Approaches:

Integration with Computational Approaches:

Advanced image analysis algorithms and machine learning approaches will increasingly enable:

• Automated tracking of ATG18/WIPI-2-positive structures

• Classification of different ATG18/WIPI-2 populations based on mobility, intensity, and morphology

• Prediction of structure-function relationships through molecular dynamics simulations

These technological advances will collectively provide unprecedented insights into ATG18/WIPI-2 dynamics, potentially revealing new aspects of its function in autophagy regulation and membrane remodeling .