ATG8H Antibody

What is ATG8H and why is it important in autophagy research?

ATG8H is one of the isoforms of the ATG8 protein family in Arabidopsis thaliana that plays essential roles in autophagy pathways. ATG8 proteins are ubiquitin-fold proteins that decorate emerging phagophores and autophagosomes following their modification with phosphatidylethanolamine (PE) . These proteins function as docking platforms for numerous autophagy adaptors and receptors that drive autophagic vesicle dynamics and cargo selection . ATG8H specifically shows notable induction in roots under phosphate-deficient conditions, suggesting its specialized role in nutrient stress responses . Understanding ATG8H function provides critical insights into plant-specific autophagy mechanisms and stress adaptation pathways.

How are ATG8H antibodies used in basic autophagy research?

ATG8H antibodies are valuable tools for detecting and quantifying ATG8H proteins in various experimental contexts. Researchers typically use these antibodies for:

• Immunoblotting to detect native ATG8H proteins and assess their abundance

• Distinguishing between lipidated (active) and non-lipidated forms of ATG8H

• Immunoprecipitation to isolate ATG8H-containing protein complexes

• Immunocytochemistry to visualize the subcellular localization of ATG8H

While ATG8H-specific antibodies can be challenging to develop due to high sequence similarity among ATG8 isoforms, polyclonal antibodies that recognize multiple ATG8 isoforms (including ATG8H) have been successfully employed in studies examining autophagy modulation under various conditions . These antibodies allow researchers to monitor autophagy activation and progression in plant systems.

What are the main differences between ATG8H and other ATG8 isoforms?

ATG8H is one of multiple ATG8 isoforms in Arabidopsis thaliana, and while these proteins share significant sequence homology, they show distinct expression patterns and potentially specialized functions:

• Expression patterns: ATG8H, along with ATG8F, is notably induced in roots under low phosphate conditions, while other isoforms may respond differently to various stresses .

• Binding specificities: Different ATG8 isoforms may have varying affinities for interaction partners. For instance, some proteins interact with ATG8 via the LIR/AIM docking site (LDS), while others use the UIM-docking site (UDS) .

• Functional redundancy: Studies with knockout mutants such as atg8h-2 and atg8h-3 have been used to investigate functional roles, though often double or multiple mutants (e.g., atg8f/atg8h) are needed to observe clear phenotypes due to redundancy .

The specific binding preferences and downstream effects of ATG8H versus other isoforms remain an active area of research, with antibodies being critical tools for distinguishing these proteins in experimental settings.

How can ATG8H antibodies be used to measure autophagic flux in plant cells?

Measuring autophagic flux is crucial for understanding the dynamics of autophagy pathways. With ATG8H antibodies, researchers can employ several approaches:

• ATG8 degradation assay: This involves treating samples with vacuolar H⁺-ATPase inhibitors like concanamycin A (Conc A) to prevent ATG8 degradation in the vacuole, then comparing ATG8 levels with and without treatment using immunoblotting with anti-ATG8 antibodies .

• Membrane association analysis: Researchers can fractionate cells into cytosolic and membrane components, then use immunoblotting to detect membrane-associated (lipidated) ATG8 proteins, which correlates with autophagic activity .

• Comparative analysis across genotypes: By comparing ATG8 protein levels between wild-type plants and autophagy-defective mutants (e.g., atg7-3), researchers can assess the impact of specific genetic backgrounds on autophagic flux .

Calculation of autophagic flux typically involves determining the ratio of accumulated ATG8 proteins after blocking degradation to the steady-state levels without inhibitors. Research has shown that this flux may vary under different conditions, such as phosphate starvation, with the atg8f/atg8h double mutant showing reduced autophagic flux under phosphate-depleted conditions compared to wild-type plants .

What are the key binding interfaces of ATG8H and how can antibodies help characterize its interactome?

ATG8H, like other ATG8 proteins, contains specific binding interfaces that mediate interactions with various proteins. Two key interfaces have been identified:

• LIR/AIM docking site (LDS): This interface recognizes the ATG8-interacting motif (AIM, also known as LC3-interacting region or LIR) with the consensus sequence W/F/Y-X-X-L/I/V .

• UIM-docking site (UDS): This newly identified interface interacts with ubiquitin-interacting motif (UIM)-like sequences, which represents an alternative binding mechanism .

Antibodies against ATG8H can be used in various approaches to characterize its interactome:

• Co-immunoprecipitation: Using ATG8H antibodies to pull down ATG8H complexes followed by mass spectrometry to identify interaction partners.

• Protein-protein interaction validation: Combined with specific mutations in either the LDS (e.g., Y50A L51A) or UDS (e.g., I77A F78A V79A) sites, researchers can determine which interface is used by specific interactors .

• In situ proximity labeling: This involves fusing ATG8H with enzymes that label proximal proteins upon activation, followed by immunoprecipitation with ATG8H antibodies.

Research has identified 112 ATG8 interactors, with 47 binding through the LDS and 19 through the UDS . These include proteins involved in autophagy regulation, cargo recognition, and vesicle dynamics.

How do researchers validate the specificity of ATG8H antibodies given the sequence similarity among ATG8 isoforms?

Validating antibody specificity for ATG8H is challenging due to the high sequence conservation among ATG8 isoforms. Researchers employ several strategies:

• Genetic validation: Testing antibody reactivity against samples from ATG8H knockout mutants (e.g., atg8h-2, atg8h-3) to confirm absence of signal .

• Recombinant protein controls: Using purified recombinant ATG8 isoforms to test cross-reactivity and establish detection thresholds.

• Peptide competition assays: Pre-incubating antibodies with ATG8H-specific peptides to block binding sites before immunoblotting or immunostaining.

• Mass spectrometry verification: Confirming the identity of immunoprecipitated proteins through peptide sequencing.

• Expression correlation: Comparing antibody signal with known expression patterns or mRNA levels of ATG8H under specific conditions, such as phosphate starvation, which induces ATG8H expression .

It's worth noting that many studies utilize polyclonal antibodies that recognize multiple ATG8 isoforms rather than strictly ATG8H-specific antibodies, due to the technical challenges in generating truly isoform-specific antibodies .

What experimental approaches can detect the ATG8H lipidation state using antibodies?

ATG8 lipidation (conjugation to phosphatidylethanolamine) is a critical step in autophagosome formation. Detecting the lipidation state of ATG8H specifically can be technically challenging but several approaches using antibodies have been developed:

• SDS-PAGE mobility shift analysis: Lipidated ATG8 (ATG8-PE) migrates faster than non-lipidated ATG8 in SDS-PAGE. By comparing migration patterns with immunoblotting, researchers can distinguish between these forms, though the small size difference makes this technically demanding .

• Urea-PAGE gel systems: These provide better separation of lipidated and non-lipidated forms for immunoblot detection.

• Membrane fractionation: Lipidated ATG8 associates with membranes, while non-lipidated forms remain cytosolic. Fractionation followed by immunoblotting helps distinguish these populations .

• Phospholipase D treatment: This enzyme cleaves the PE moiety from ATG8-PE, causing a mobility shift that can be detected by immunoblotting.

• Detergent resistance: Lipidated ATG8 shows differential solubility in certain detergents, which can be leveraged for separation before immunodetection.

Research has shown that membrane-associated ATG8s (likely representing lipidated forms) are substantially decreased in autophagy-defective mutants like atg7-3 and also reduced in ATG8 isoform-specific mutants like atg8f/atg8h under phosphate-depleted conditions .

What are the optimal fixation and immunostaining protocols for detecting ATG8H in plant tissues?

For successful immunolocalization of ATG8H in plant tissues, researchers should consider the following protocols:

• Fixation options:

  • Aldehyde fixation (4% paraformaldehyde) preserves protein antigenicity while maintaining cellular structure

  • For better membrane preservation, a combination of paraformaldehyde and glutaraldehyde (0.1-0.5%) may be used

  • Cold methanol fixation can be beneficial for visualizing membrane-associated ATG8

• Permeabilization:

  • Plant cell walls require additional permeabilization steps

  • Enzymatic digestion (cellulase/macerozyme) or mechanical sectioning prior to antibody incubation

  • Detergent treatment (0.1-0.5% Triton X-100) to permeabilize membranes

• Antigen retrieval:

  • Heat-induced epitope retrieval may be necessary if aldehyde fixation causes epitope masking

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) are commonly used

• Blocking and antibody dilutions:

  • 3-5% BSA or normal serum from the secondary antibody host species

  • Primary antibody dilutions typically range from 1:100 to 1:1000 depending on antibody quality

  • Extended incubation times (overnight at 4°C) often yield better results with plant tissues

• Controls:

  • Include atg8h mutant tissues as negative controls

  • Compare patterns with autophagy-deficient mutants (e.g., atg7-3)

  • Use competing peptides to verify specificity

The morphology of ATG8H-positive structures changes during autophagy progression, from cytosolic puncta (representing early autophagosomes) to vacuolar structures in later stages, particularly under stress conditions such as phosphate starvation that induce ATG8H expression .

How can researchers quantitatively analyze autophagy using ATG8H antibodies in different experimental systems?

Quantitative analysis of autophagy using ATG8H antibodies requires well-established approaches:

• Western blotting quantification:

  • Measure the ratio of lipidated to non-lipidated ATG8H (though technically challenging)

  • Compare total ATG8 levels with and without autophagy inhibitors like concanamycin A to determine flux

  • Standardize using reference proteins (e.g., actin, tubulin) for normalization

• Immunofluorescence quantification:

  • Count ATG8H-positive puncta per cell area

  • Measure size distribution of ATG8H-positive structures

  • Track colocalization with other autophagy markers (e.g., ATG5, ATG9)

• Biochemical fractionation analysis:

  • Quantify membrane-associated ATG8 levels in microsomal fractions

  • Compare cytosolic versus membrane-bound ATG8 ratios

• Flux measurement approaches:

  • Calculate the accumulation of ATG8 after treatment with lysosomal/vacuolar inhibitors

  • Use the formula: Flux = (ATG8 with inhibitor - ATG8 without inhibitor)/ATG8 without inhibitor

*Data reconstructed based on research findings in search result , showing reduced autophagic flux in atg8f/atg8h mutants under Pi-depleted conditions.

What are the pitfalls and troubleshooting approaches when using ATG8H antibodies in autophagy research?

When working with ATG8H antibodies, researchers should be aware of several common challenges and their solutions:

• Cross-reactivity issues:

  • Challenge: ATG8 isoforms share high sequence similarity, making specific detection difficult.

  • Solution: Validate using knockout mutants (atg8h-2, atg8h-3) , perform peptide competition assays, or use recombinant protein controls.

• Lipidation state discrimination:

  • Challenge: It can be difficult to distinguish lipidated and non-lipidated forms on standard gels.

  • Solution: Utilize urea-containing gels, optimize running conditions, or perform membrane fractionation before analysis .

• Background signals:

  • Challenge: High background can obscure specific signals, especially in plant tissues.

  • Solution: Optimize blocking conditions, use longer wash steps, and purify antibodies if necessary.

• Fixation artifacts:

  • Challenge: Fixation can alter epitope accessibility or create false structures.

  • Solution: Compare multiple fixation methods and validate with live-cell imaging approaches when possible.

• Autophagic flux interpretation:

  • Challenge: Changes in ATG8 levels could reflect altered synthesis rather than autophagy.

  • Solution: Always include flux measurements with lysosomal/vacuolar inhibitors like concanamycin A .

• Technical variability:

  • Challenge: Inconsistent results between experiments.

  • Solution: Standardize sample preparation, include positive and negative controls in each experiment, and use internal loading controls.

• Physiological versus basal autophagy:

  • Challenge: Distinguishing stress-induced from constitutive autophagy.

  • Solution: Include appropriate stress and non-stress conditions, and compare wild-type with autophagy-deficient mutants .

How can ATG8H antibodies help identify novel specific interactors of this isoform versus other ATG8 variants?

To identify ATG8H-specific interactors, researchers can implement several sophisticated approaches using antibodies:

• Comparative immunoprecipitation: Performing parallel immunoprecipitations with antibodies against different ATG8 isoforms followed by mass spectrometry can reveal isoform-specific binding partners. For ATG8H, this might identify proteins that preferentially interact with this isoform, particularly under phosphate starvation conditions .

• Proximity-dependent labeling: Techniques like BioID or APEX2 fusion with ATG8H can identify proteins in close proximity in vivo, which can then be validated with co-immunoprecipitation using ATG8H antibodies.

• Domain-focused approaches: By comparing binding to wild-type ATG8H versus mutated versions with alterations in the LDS (Y50A L51A) or UDS (I77A F78A V79A) binding surfaces, researchers can determine which interface mediates specific interactions .

• Conditional interaction screening: Exposing plants to different stresses (particularly phosphate starvation) before immunoprecipitation may reveal condition-specific interactors of ATG8H .

Research using similar approaches has identified 112 ATG8e interactors, with 47 binding via the LDS and 19 via the UDS . For ATG8H, studies suggest it may have specialized functions during phosphate starvation, potentially through unique binding partners that regulate root responses to nutrient limitation .

What is the role of ATG8H in selective autophagy pathways, and how can antibodies help elucidate these functions?

ATG8H, like other ATG8 isoforms, likely participates in selective autophagy pathways, targeting specific cellular components for degradation. Antibodies can help elucidate these functions through:

• Co-localization studies: Using ATG8H antibodies alongside markers for specific organelles or cellular structures can identify which components are being targeted under different conditions.

• Cargo identification: Immunoprecipitating ATG8H-positive autophagosomes followed by proteomics can identify selective cargo.

• Autophagy receptor interactions: Analyzing which selective autophagy receptors (like NBR1, DSK2, or ATI1/2) preferentially interact with ATG8H versus other isoforms.

• Mutant complementation studies: Testing whether reintroducing ATG8H can restore specific selective autophagy pathways in atg8h mutants.

Available research suggests that ATG8 proteins interact with numerous receptors for unwanted or dysfunctional cellular components, including mitochondria, peroxisomes, ER, lipid droplets, ribosomes, proteasomes, and pathogens . ATG8H specifically shows increased expression under phosphate starvation , suggesting it may have specialized roles in phosphate homeostasis, potentially through selective degradation of phosphate-containing macromolecules or organelles.

How do post-translational modifications affect ATG8H function, and how can antibodies detect these modifications?

Post-translational modifications (PTMs) of ATG8H can significantly alter its function in autophagy pathways. To investigate these PTMs:

• Modification-specific antibodies: Researchers can develop antibodies that specifically recognize modified forms of ATG8H, such as:

  • Phosphorylated ATG8H

  • Acetylated ATG8H

  • Ubiquitinated ATG8H

• Two-dimensional gel electrophoresis: Combining isoelectric focusing with SDS-PAGE followed by immunoblotting with ATG8H antibodies can separate differently modified forms.

• Mass spectrometry after immunoprecipitation: Using ATG8H antibodies to purify the protein followed by mass spectrometry to identify PTM sites and types.

• In vitro modification assays: Testing how specific enzymes modify recombinant ATG8H, then detecting these changes with general ATG8H antibodies.

While the core regulatory mechanism for ATG8 proteins involves lipidation with PE through an ATP-dependent conjugation cascade involving ATG7 (E1), ATG3 (E2), and the ATG12-ATG5-ATG16 complex (E3) , additional PTMs likely provide another layer of regulation. These modifications may influence ATG8H's binding affinities for specific interaction partners, subcellular localization, or stability, potentially explaining its specialized functions under conditions like phosphate starvation .

How can ATG8H antibodies be used to study the relationship between autophagy and nutrient stress responses?

ATG8H shows notable induction under phosphate starvation conditions , suggesting a specialized role in nutrient stress responses. Researchers can use ATG8H antibodies to investigate these connections through:

• Nutrient-dependent expression profiling: Quantifying ATG8H protein levels under various nutrient stresses (phosphate, nitrogen, carbon, sulfur limitation) using immunoblotting.

• Stress-induced relocalization: Tracking changes in ATG8H localization during nutrient stress using immunofluorescence.

• Selective nutrient-dependent autophagy: Identifying whether ATG8H preferentially associates with specific organelles or structures during nutrient limitation.

• Comparative studies with mutants: Analyzing how atg8h mutants respond to nutrient stress compared to wild-type plants using antibodies to track autophagy markers.

Studies have shown that the autophagic flux in atg8f/atg8h double mutants is specifically reduced under phosphate-depleted conditions compared to wild-type plants , suggesting these isoforms play critical roles in phosphate starvation responses. A comprehensive experimental approach combining ATG8H antibody detection with physiological and biochemical analyses can help elucidate the mechanisms by which ATG8H-mediated autophagy contributes to nutrient adaptation.

What are the latest methodological advances in using ATG8H antibodies for live-cell imaging and dynamic studies?

While traditional antibodies cannot access intracellular proteins in living cells, several innovative approaches now allow researchers to study ATG8H dynamics:

• Intrabodies: Engineered antibody fragments (e.g., nanobodies, scFvs) that recognize ATG8H can be expressed intracellularly and fused to fluorescent proteins for live imaging.

• Modified cell-penetrating antibodies: Chemical modifications or peptide conjugation can enable antibodies to cross membranes for live-cell applications.

• Split fluorescent protein complementation: By fusing half of a fluorescent protein to an ATG8H-binding peptide and the other half to a potential interaction partner, researchers can visualize ATG8H interactions in living cells.

• FRET/FLIM with tagged binding partners: Studying energy transfer between fluorescently-labeled ATG8H-binding proteins and potential partners.

• Correlative microscopy approaches: Combining live imaging with subsequent immunostaining using ATG8H antibodies on the same sample to correlate dynamic events with molecular identities.