WDR41 Antibody

What is WDR41 and what cellular functions does it perform?

WDR41 is a WD40-repeat containing protein that serves as a non-catalytic component of the C9orf72-SMCR8 complex, which has guanine nucleotide exchange factor (GEF) activity and regulates autophagy . The protein consists of 459 amino acids with a molecular weight of approximately 52 kDa .

The primary functions of WDR41 include:

• Recruitment of C9orf72 and SMCR8 to lysosomes, particularly under starvation conditions

• Regulation of autophagy through its association with the C9orf72-SMCR8 complex

• Involvement in membrane trafficking and the autophagy-lysosome pathway

• Participation in the acute activation of mechanistic target of rapamycin complex 1 (mTORC1) by amino acids

Notably, WDR41 localizes to lysosomes, especially during cellular starvation, and is required for the recruitment of its binding partners to these organelles . This localization is independent of changes in mTORC1 or Unc-51-like kinase (ULK) complex signaling and does not depend on autophagy .

What are the recommended applications for WDR41 antibodies in research?

Based on available commercial antibodies and research protocols, WDR41 antibodies are suitable for multiple experimental applications:

When designing experiments, researchers should consider that WDR41 displays a diffuse localization pattern in multiple cell types under normal conditions but shows enriched lysosomal localization during starvation . Therefore, immunofluorescence studies should include appropriate controls and starvation conditions to observe the dynamic localization of WDR41.

How should WDR41 antibodies be validated before experimental use?

Thorough validation of WDR41 antibodies is crucial for generating reliable results. Recommended validation approaches include:

• Western blot analysis with positive and negative controls:

  • Use fetal human brain tissue as a positive control, as it shows detectable WDR41 expression

  • Include WDR41 knockout (KO) cell lines as negative controls

  • Verify the observed molecular weight (approximately 52 kDa)

• Immunofluorescence specificity testing:

  • Compare staining patterns in wild-type versus WDR41 KO cells

  • Include co-localization studies with lysosomal markers like LAMP1 during starvation

• Epitope verification:

  • Check immunogen information to ensure the antibody targets a unique region of WDR41

  • Consider using antibodies targeting different epitopes to confirm results

• Cross-reactivity assessment:

  • Test reactivity in multiple species if cross-species studies are planned

  • Evaluate potential cross-reactivity with other WD40-repeat containing proteins

Research by Amick et al. demonstrated successful CRISPR-Cas9 generation of WDR41 knockout cell lines, which provide excellent negative controls for antibody validation .

How can WDR41 antibodies be used to study the C9orf72-SMCR8-WDR41 complex in autophagy regulation?

The C9orf72-SMCR8-WDR41 complex plays important roles in autophagy regulation, and WDR41 antibodies can be instrumental in dissecting these mechanisms:

• Co-immunoprecipitation studies:

  • WDR41 antibodies can be used to pull down the entire complex and identify additional interacting partners

  • Research has shown that WDR41 interacts directly with C9orf72 even in the absence of SMCR8, suggesting it may serve as a scaffold for complex formation

• Lysosomal recruitment analysis:

  • Immunofluorescence with WDR41 antibodies can track the recruitment of the complex to lysosomes under different conditions

  • This can be particularly valuable when combined with lysosomal isolation techniques, such as iron dextran nanoparticle-based lysosome purification

• GEF activity assays:

  • The complex promotes the exchange of GDP to GTP on RAB8A and RAB39B

  • WDR41 antibodies can be used in depletion studies to determine how complex composition affects this activity

• Autophagy flux measurements:

  • Combined with LC3 lipidation analysis, WDR41 antibodies can help determine if manipulation of the complex alters autophagy initiation or progression

  • WDR41 knockout cells show no significant defect in LC3 lipidation during starvation, suggesting WDR41 is not essential for autophagy induction

Importantly, research shows that the C9orf72-SMCR8-WDR41 complex acts as a negative regulator of autophagy initiation by interacting with the ULK1/ATG1 kinase complex and inhibiting its protein kinase activity .

What experimental approaches can distinguish between different cellular localizations of WDR41?

WDR41 shows dynamic subcellular localization, and several experimental approaches can help characterize these patterns:

• Endogenous tagging strategies:

  • CRISPR/Cas9-mediated insertion of epitope tags (e.g., 2xHA) at the C-terminus of the endogenous WDR41 gene avoids overexpression artifacts

  • This approach revealed diffuse localization under fed conditions but pronounced lysosomal localization during starvation

• Subcellular fractionation combined with western blotting:

  • Magnetic isolation of lysosomes using iron dextran nanoparticles achieved approximately 40-fold enrichment of lysosomal proteins

  • This technique confirmed increased WDR41 association with lysosomes during starvation

• Co-localization analysis with compartment-specific markers:

  • LAMP1 co-staining for lysosomes

  • Previous studies incorrectly identified WDR41 as Golgi-localized based on overexpression studies, highlighting the importance of studying endogenously expressed protein

• Live-cell imaging:

  • Enables real-time monitoring of WDR41 recruitment to lysosomes in response to nutrient changes

  • Can be combined with treatments that affect autophagy or lysosomal function

Research demonstrated that WDR41 lysosomal localization increases during starvation but is independent of changes in mTORC1 or ULK complex signaling and does not require autophagy .

How should researchers design experiments to study WDR41 in the context of cancer progression?

WDR41 shows altered expression in cancer, particularly in breast cancer, suggesting potential tumor suppressor functions. Researchers should consider these approaches:

• Expression analysis in cancer versus normal tissues:

  • WDR41 is expressed at low levels in breast cancer, especially in triple-negative breast cancer (TNBC)

  • Immunohistochemical staining shows higher expression in para-carcinoma tissues than in paired tumor tissues

  • Western blot analysis revealed reduced WDR41 levels in approximately 78.6% (11/14) of tumor samples compared to paired normal breast tissue

• Epigenetic regulation studies:

  • Methylation-specific PCR (MSP) can detect WDR41 hypermethylation in cancer cell lines

  • Treatment with methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) increases WDR41 expression in MDA-MB-231 cells (TNBC) but not in MCF-10A (normal mammary epithelial cells) or estrogen receptor-positive MCF-7 cells

• Functional studies using gain/loss-of-function approaches:

  • WDR41 down-regulation promotes tumor characteristics (cell viability, cell cycle progression, migration) in TNBC cells

  • WDR41 up-regulation inhibits these processes and suppresses tumor growth in vivo

• Signaling pathway analysis:

  • WDR41 ablation activates the AKT/GSK-3β pathway and subsequent nuclear activation of β-catenin in MDA-MB-231 cells

  • AKT inhibitor MK-2206 prevents WDR41 down-regulation-mediated activation of GSK-3β/β-catenin signaling

These findings suggest that methylated WDR41 in TNBC cells promotes tumorigenesis through positive regulation of the AKT/GSK-3β/β-catenin pathway, providing a potential therapeutic target .

What are the optimal fixation and permeabilization methods for WDR41 immunofluorescence studies?

When performing immunofluorescence studies with WDR41 antibodies, researchers should consider these methodological factors:

• Fixation protocol:

  • Paraformaldehyde (4%) fixation for 15-20 minutes at room temperature preserves protein localization while maintaining antigen accessibility

  • Avoid methanol fixation as it can disrupt protein-protein interactions and affect the detection of complexes

• Permeabilization approach:

  • Gentle permeabilization with 0.1-0.2% Triton X-100 for 5-10 minutes is typically sufficient

  • For detailed membrane structure analysis, consider milder detergents like 0.1% saponin

• Blocking conditions:

  • Use 5% normal serum (from the species of the secondary antibody) with 1% BSA to reduce background

  • Include 0.1% Triton X-100 in blocking solution to maintain permeabilization

• Antibody incubation:

  • Overnight incubation at 4°C with primary antibodies at dilutions of 1:100-1:500

  • Secondary antibody incubation for 1-2 hours at room temperature

• Special considerations:

  • Include physiological manipulations (e.g., starvation) to observe dynamic WDR41 localization

  • Co-stain with LAMP1 or other lysosomal markers to confirm lysosomal localization

Research has shown that endogenously tagged WDR41 (WDR41-2xHA) displays diffuse staining under normal conditions but co-localizes with LAMP1 during starvation .

How should researchers interpret conflicting data between WDR41 mRNA and protein levels?

Reconciling discrepancies between WDR41 mRNA and protein levels requires careful experimental design and interpretation:

• Comprehensive analysis approach:

  • Compare mRNA levels (qRT-PCR) with protein expression (Western blot, immunohistochemistry) in the same samples

  • In breast cancer studies, both mRNA and protein levels of WDR41 were decreased in cancer cells compared to normal cells, but the magnitude of reduction varied

• Post-transcriptional regulation assessment:

  • Investigate protein stability using cycloheximide chase assays

  • Examine potential microRNA-mediated regulation of WDR41 translation

• Context-dependent regulation:

  • Consider cell type-specific factors that might affect WDR41 expression

  • The degree of WDR41 mRNA reduction correlates with cell invasiveness (MDA-MB-231: 50% reduction; SKBR3: 75% reduction; MCF-7: 30% reduction)

• Epigenetic regulation:

  • Assess DNA methylation status of the WDR41 promoter using methylation-specific PCR

  • Treatment with demethylating agents (5-aza-dC) can help determine if methylation affects WDR41 expression

• Methodological validation:

  • Use multiple antibodies targeting different epitopes of WDR41

  • Include WDR41 knockout cells as negative controls in protein analysis

Studies in breast cancer have shown that while both mRNA and protein levels of WDR41 are reduced, epigenetic mechanisms (particularly DNA methylation) play a significant role in regulating WDR41 expression .

What controls should be included when studying WDR41 in lysosomal localization experiments?

Rigorous controls are essential for accurate interpretation of WDR41 lysosomal localization studies:

• Genetic controls:

  • Include WDR41 knockout cell lines as negative controls

  • Use cells expressing endogenously tagged WDR41 (e.g., WDR41-2xHA) to avoid overexpression artifacts

• Physiological condition controls:

  • Compare fed versus starved conditions, as WDR41 shows enhanced lysosomal localization during starvation

  • Include time course experiments to capture dynamic recruitment

• Pathway manipulation controls:

  • Test treatments that affect mTORC1 signaling (e.g., torin 1) or autophagy (e.g., concanamycin A)

  • Research has shown that WDR41 recruitment to lysosomes is independent of changes in mTORC1 or ULK complex signaling

• Co-localization markers:

  • Include LAMP1 staining as a standard lysosomal marker

  • Consider multiple lysosomal markers to confirm specificity

• Technical validation:

  • Use both immunofluorescence and biochemical fractionation (lysosome isolation) to confirm localization

  • Magnetic isolation of lysosomes using iron dextran nanoparticles provides approximately 40-fold enrichment of lysosomal proteins

Research demonstrated that WDR41 is essential for recruiting C9orf72 and SMCR8 to lysosomes, especially during starvation, and this function is independent of autophagy induction .

What are the technical considerations for studying WDR41 in the context of autophagy?

When investigating WDR41's role in autophagy regulation, researchers should consider these methodological approaches:

• Autophagy flux measurements:

  • Monitor LC3 lipidation (LC3-I to LC3-II conversion) with and without lysosomal inhibitors (e.g., concanamycin A)

  • Research shows WDR41 knockout cells do not have significant defects in LC3 lipidation during starvation

• Protein interaction studies:

  • Investigate WDR41's interactions with autophagy-related proteins using co-immunoprecipitation

  • The C9orf72-SMCR8-WDR41 complex interacts with the ULK1/ATG1 kinase complex and inhibits its activity

• Subcellular localization analysis:

  • Examine WDR41 co-localization with autophagosome markers (e.g., LC3) under different conditions

  • WDR41 recruitment to lysosomes does not reflect a general response to increased autophagosome abundance

• Genetic manipulation approaches:

  • Compare autophagy in wild-type versus WDR41 knockout cells

  • Express lysosome-targeted versions of complex components to bypass WDR41 requirement

• Nutrient sensing experiments:

  • Monitor amino acid-induced mTORC1 activation in WDR41-deficient cells

  • WDR41 knockout cells show defects in acute activation of mTORC1 by amino acids

Research has demonstrated that while the C9orf72-SMCR8-WDR41 complex has been implicated in autophagy, WDR41 itself is not essential for autophagy induction, suggesting more complex regulatory roles .

How can WDR41 antibodies contribute to research on neurodegenerative diseases?

WDR41 has been associated with neurodegenerative conditions, particularly frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) . WDR41 antibodies enable several research approaches in this context:

• Expression analysis in disease models:

  • Compare WDR41 levels in normal versus diseased tissue samples

  • Examine expression patterns in neurons, glia, and other relevant cell types

• C9orf72-related mechanism studies:

  • Mutations in C9orf72 are a major genetic cause of ALS and FTD

  • WDR41 antibodies can help elucidate how these mutations affect formation and function of the C9orf72-SMCR8-WDR41 complex

• Autophagy dysfunction analysis:

  • Neurodegenerative diseases often involve impaired autophagy

  • Investigate how disease-related mutations affect WDR41's role in autophagy regulation

• Therapeutic target validation:

  • Assess whether modulating WDR41 levels or its interactions could restore disrupted cellular processes

  • Monitor changes in WDR41 localization in response to therapeutic interventions

• Biomarker development:

  • Explore whether altered WDR41 expression or localization correlates with disease progression

  • Investigate potential for diagnostic applications

The involvement of WDR41 in the C9orf72-SMCR8 complex, which regulates membrane trafficking and the autophagy-lysosome pathway, positions it as a valuable target for understanding pathogenic mechanisms in neurodegenerative diseases .

What methodological approaches are recommended for studying WDR41 methylation in cancer research?

Based on findings that WDR41 methylation status affects cancer progression , researchers should consider these methodological approaches:

• Methylation detection techniques:

  • Methylation-specific PCR (MSP) for targeted analysis of WDR41 promoter methylation

  • Bisulfite sequencing for comprehensive CpG methylation profiling

  • Methylation arrays for genome-wide context

• Functional validation of methylation effects:

  • Treatment with demethylating agents (e.g., 5-aza-dC) to assess expression restoration

  • Luciferase reporter assays with methylated versus unmethylated WDR41 promoter regions

• Clinical correlation studies:

  • Compare WDR41 methylation status across cancer subtypes

  • Analyze associations between methylation, expression levels, and patient outcomes

  • Research shows particularly low WDR41 expression in triple-negative breast cancer

• Mechanistic pathway investigations:

  • Examine how WDR41 methylation affects the AKT/GSK-3β/β-catenin pathway

  • Use pathway inhibitors (e.g., MK-2206 for AKT inhibition) to validate mechanistic connections

• In vivo validation:

  • Xenograft models comparing tumors with normal versus altered WDR41 expression

  • WDR41 up-regulation dramatically suppresses tumor growth in vivo

Research has demonstrated that methylated WDR41 in MDA-MB-231 cells promotes tumorigenesis through positively regulating the AKT/GSK-3β/β-catenin pathway, providing an important foundation for treating triple-negative breast cancer .

What are the common challenges in detecting endogenous WDR41 protein and how can they be addressed?

Researchers may encounter several challenges when detecting endogenous WDR41:

• Low expression levels:

  • Use sensitive detection methods such as enhanced chemiluminescence (ECL) or fluorescent secondary antibodies

  • Consider concentration of the protein through immunoprecipitation before western blotting

  • WDR41 is expressed at low levels in certain tissues and cancer types

• Antibody specificity issues:

  • Validate antibodies using WDR41 knockout cells as negative controls

  • Use epitope-tagged WDR41 (e.g., WDR41-2xHA) as positive controls

  • Consider multiple antibodies targeting different epitopes

• Background signal in immunohistochemistry/immunofluorescence:

  • Optimize blocking conditions (5% serum, 1% BSA)

  • Increase washing steps and duration

  • Use monoclonal antibodies for higher specificity

• Dynamic localization:

  • Include appropriate physiological conditions (e.g., starvation) to observe expected localization patterns

  • WDR41 shows diffuse localization under normal conditions but enriched lysosomal localization during starvation

• Protein degradation during sample preparation:

  • Include protease inhibitors in all buffers

  • Maintain samples at 4°C during processing

  • Consider gentler lysis conditions for maintaining protein complexes

Research has shown that endogenous tagging strategies using CRISPR/Cas9-mediated genome editing to insert epitope tags (2xHA) at the C-terminus of WDR41 can facilitate detection while maintaining physiological expression levels .

How can researchers optimize co-immunoprecipitation protocols for studying WDR41-containing protein complexes?

When investigating WDR41's interactions within the C9orf72-SMCR8 complex or with other proteins, consider these optimization strategies:

• Lysis buffer composition:

  • Use gentle, non-denaturing buffers (e.g., RIPA or NP-40-based)

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

  • Adjust salt concentration (150-300 mM NaCl) to balance specificity and recovery

• Antibody selection and validation:

  • Test multiple WDR41 antibodies for immunoprecipitation efficiency

  • Consider epitope-tagged versions (e.g., GFP-WDR41) for complex studies

  • Research shows WDR41 copurifies with GFP-C9orf72 even in the absence of SMCR8

• Crosslinking approaches:

  • Consider reversible crosslinkers for stabilizing transient interactions

  • Optimize crosslinking time and concentration to avoid non-specific aggregation

• Complex assessment strategies:

  • Investigate interactions reciprocally (pull down with anti-WDR41 vs. anti-C9orf72 vs. anti-SMCR8)

  • Include knockout controls for each component to assess requirement for complex formation

  • Studies show C9orf72 is the major determinant of WDR41 incorporation into the heterotrimeric complex

• Condition-dependent interactions:

  • Compare interactions under fed versus starved conditions

  • Test effects of mTOR inhibitors or autophagy modulators on complex formation

Research demonstrated that while WDR41 can interact with C9orf72 in the absence of SMCR8, no interaction was detected between SMCR8 and WDR41 in the absence of C9orf72, supporting a model where C9orf72 mediates WDR41 incorporation into the complex .

What strategies can resolve contradictory findings regarding WDR41 subcellular localization?

Previous studies have reported different subcellular localizations for WDR41, including Golgi and lysosomal distributions. These contradictions can be resolved through:

• Endogenous versus overexpression systems:

  • Overexpressed GFP-tagged WDR41 was previously reported to localize to the Golgi

  • Studies of endogenously expressed WDR41 (via 2xHA tagging) showed primarily lysosomal localization during starvation

  • Always compare overexpression results with endogenous protein localization

• Physiological condition considerations:

  • WDR41 shows diffuse localization under fed conditions but enriched lysosomal localization during starvation

  • Include multiple physiological conditions in all localization studies

• Complementary methodological approaches:

  • Combine immunofluorescence with subcellular fractionation and western blotting

  • Magnetic isolation of lysosomes using iron dextran nanoparticles confirmed WDR41 lysosomal association

• Multi-marker co-localization analysis:

  • Use multiple organelle markers simultaneously (e.g., LAMP1 for lysosomes, GM130 for Golgi)

  • Quantify co-localization coefficients for objective comparison

• Super-resolution microscopy:

  • Apply techniques like STED or STORM for more precise localization

  • This can help distinguish between closely apposed organelles