mtmr8 Antibody

What is MTMR8 and why is it an important research target?

MTMR8 (Myotubularin Related Protein 8) is a member of the myotubularin-related family of phosphatase enzymes involved in the regulation of cell growth, differentiation, and survival. It belongs to a subfamily that includes MTMR6 and MTMR7, all of which dimerize with the catalytically inactive MTMR9 . MTMR8 functions as a phosphatase that acts on lipids with phosphoinositol headgroups, specifically dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] at the D-3 position . The importance of studying MTMR8 stems from its role in multiple cellular processes including autophagy regulation, actin cytoskeleton organization, and muscle development . Additionally, dysregulation of MTMR8 has been implicated in various diseases, including cancer and neurodegenerative disorders , making it a significant target for researchers investigating these conditions.

What are the key applications for MTMR8 antibodies in research?

MTMR8 antibodies serve multiple critical applications in research:

These applications enable researchers to investigate MTMR8 expression patterns, protein-protein interactions, and subcellular localization. When studying MTMR8's role in autophagy, researchers commonly employ Western blotting to detect changes in LC3B-II and p62 levels following MTMR8 depletion . For examining MTMR8's involvement in muscle development, immunohistochemistry is particularly valuable for visualizing protein distribution in tissue sections .

What is the reported molecular weight of MTMR8 and why might observed weights differ?

• Proteintech reports an observed molecular weight of 55-58 kDa for their MTMR8 antibody (12299-1-AP)

• Abbexa reports a calculated molecular weight of 63.5 kDa for human MTMR8

These discrepancies may result from:

• Post-translational modifications affecting protein migration

• Detection of different isoforms (up to 2 different isoforms have been reported)

• Proteolytic processing during sample preparation

• Antibody specificity for different epitopes or domains

When validating a new MTMR8 antibody, researchers should run appropriate positive controls such as HeLa cells, A375 cells, or human placenta tissue, which have been confirmed to express MTMR8 .

How should researchers design experiments to study MTMR8-MTMR9 interactions?

To effectively study MTMR8-MTMR9 interactions, researchers should implement comprehensive experimental approaches:

• Co-immunoprecipitation studies: Express HA-tagged MTMR8 and FLAG-tagged MTMR9 in cells (e.g., HeLa), then immunoprecipitate using anti-HA and anti-FLAG antibodies. Both proteins should be detected when either is immunoprecipitated, indicating complex formation .

• Protein stability analyses: Treat cells co-expressing MTMR8 and MTMR9 with cycloheximide to inhibit protein synthesis, then monitor MTMR8 degradation rates in the presence versus absence of MTMR9. Higher MTMR8 levels and slower degradation in cells co-expressing both proteins would suggest complex formation increases stability .

• Enzymatic activity assays: Measure phosphatase activity toward PtdIns(3)P and PtdIns(3,5)P₂ substrates using purified proteins. Compare activity of MTMR8 alone versus MTMR8-MTMR9 complex, as MTMR9 increases MTMR8's activity 4-fold toward PtdIns(3)P and 1.4-fold toward PtdIns(3,5)P₂ .

• Complex dissociation studies: Place cells expressing HA-MTMR8 and endogenous MTMR9 in serum-free medium and monitor complex stability over time. The complex typically dissociates completely by 2 hours in serum-starvation conditions .

When reporting results, researchers should include data from multiple approaches to provide comprehensive evidence of the interaction and its functional significance.

What are the optimal methods for knockdown studies of MTMR8, and what controls should be included?

For effective MTMR8 knockdown studies, researchers should consider the following methodological approaches:

• Morpholino design: Two approaches have proven effective:

  • Splice junction morpholino targeting the first coding exon-intron boundary (MO1) to introduce a premature termination codon

  • Splice junction morpholino targeting the second coding exon-intron boundary (MO2) to eliminate the exon encoding part of the PH/G domain

• siRNA knockdown: For mammalian cells, researchers should:

  • Design siRNAs targeting conserved regions of MTMR8

  • Validate knockdown efficiency using qRT-PCR (target reduction to <30% of control levels)

  • Confirm knockdown at protein level using Western blot

• Essential controls:

  • Control morpholino or non-targeting siRNA

  • Phenotype rescue with morpholino/siRNA-resistant MTMR8 construct

  • Combined knockdown of MTMR8 and MTMR9 to evaluate functional interactions

  • Bafilomycin A1 treatment (100 nM for 3 hours) when studying autophagy to inhibit fusion between autophagosomes and lysosomes

• Knockdown validation parameters:

  • RT-PCR to verify altered transcript (for morpholinos)

  • qRT-PCR to quantify knockdown efficiency (target: MTMR8 level <0.3 relative to control siRNA)

  • Western blot to confirm protein reduction

Studies have shown that knockdown of MTMR8 alone may have limited effects on some processes (e.g., autophagy), while combined knockdown with MTMR9 produces more pronounced phenotypes, suggesting functional redundancy or compensatory mechanisms .

How can researchers effectively study MTMR8's role in autophagy?

To effectively investigate MTMR8's role in autophagy, researchers should employ a multi-faceted experimental approach:

• Autophagy flux assessment:

  • Monitor LC3B-II and p62 levels by Western blot in MTMR8-depleted cells under both basal and serum-starved conditions

  • Include Bafilomycin A1 treatment (100 nM for 3 hours) to block autophagosome-lysosome fusion, allowing accumulation of autophagosomes

  • A further increase in LC3B-II and p62 levels after Bafilomycin A1 treatment in MTMR8-depleted cells indicates impaired autophagic flux rather than blocked autophagosome formation

• PtdIns(3)P monitoring:

  • Use immunofluorescence with antibodies specifically recognizing PtdIns(3)P

  • Count PtdIns(3)P-positive structures (spots larger than 1 nm) in control versus MTMR8-depleted or MTMR8-overexpressing cells

  • Include MTMR8+MTMR9 co-expression, as this combination significantly alters cellular PtdIns(3)P levels

• TFEB nuclear translocation:

  • Examine TFEB localization by immunofluorescence in MTMR8-depleted cells

  • Compare serum-fed versus serum-starved conditions

  • MTMR8-depleted cells display nuclear TFEB in both conditions, suggesting constitutive activation of autophagy-lysosome gene expression

• Combined knockdown approach:

  • Compare effects of MTMR8 knockdown alone versus MTMR8+MTMR9 double knockdown

  • Only the combined knockdown significantly reduces p62 levels in Bafilomycin A1-treated cells

This comprehensive approach allows researchers to distinguish between effects on autophagosome formation versus degradation and to understand the relationship between MTMR8's phosphatase activity and autophagic processes.

Why might researchers observe different results when studying MTMR8's effects on PtdIns(3)P versus PtdIns(3,5)P₂ levels?

Researchers may encounter discrepancies when studying MTMR8's effects on different phosphoinositide substrates due to several methodological and biological factors:

When interpreting contradictory results, researchers should consider these factors and integrate findings from both in vitro enzymatic assays and cellular studies to develop a complete understanding of MTMR8's phosphatase activity in physiological contexts.

How should researchers interpret contradictory findings regarding MTMR8's function in different model systems?

When faced with contradictory findings regarding MTMR8's function across different model systems, researchers should employ a systematic interpretive framework:

• Species-specific differences:

  • MTMR8 orthologs have been identified in human, mouse, rat, frog, zebrafish, chimpanzee, and chicken

  • Zebrafish studies show MTMR8 is predominantly expressed in eye field and somites during early somitogenesis

  • Human studies focus on MTMR8's role in autophagy regulation and interaction with MTMR9

  • These differences may reflect evolutionary adaptations or tissue-specific functions

• Context-dependent functions:

  • MTMR8 works in a non-cell autonomous manner in actin modeling according to cell transplantation experiments

  • The protein's function may depend on the presence of interacting partners like MTMR9

  • Environmental conditions (e.g., serum starvation) affect MTMR8-MTMR9 complex stability

• Methodological variations:

  • Loss-of-function through morpholino knockdown in zebrafish versus siRNA in mammalian cells

  • Complete ablation versus partial reduction of protein levels

  • Acute versus chronic protein depletion

• Integration framework:

  • Construct a unified model incorporating tissue-specific, developmental stage-specific, and condition-specific functions

  • Example: MTMR8's role in actin modeling during development may use the same enzymatic activity that regulates autophagy in adult tissues

  • Pathway analysis reveals MTMR8 participates in both Hedgehog signaling and autophagy regulation , suggesting multifunctional capabilities

When publishing results, researchers should explicitly discuss model system limitations and potential reasons for discrepancies with existing literature, enhancing the field's collective understanding of MTMR8's diverse functions.

What are common pitfalls in MTMR8 antibody-based experiments and how can they be avoided?

Researchers working with MTMR8 antibodies should be aware of several common pitfalls and implement appropriate strategies to overcome them:

• Antibody specificity issues:

  • Problem: Cross-reactivity with other MTMR family members due to sequence homology

  • Solution: Validate antibody specificity using MTMR8 knockdown/knockout samples and Western blot analysis across multiple cell lines (HeLa, A375, and human placenta tissue are recommended positive controls)

• Detection of unexpected molecular weights:

  • Problem: Observed molecular weights (55-58 kDa) often differ from calculated weight (79 kDa)

  • Solution: Include positive controls with confirmed MTMR8 expression; test multiple antibodies targeting different epitopes; consider the presence of isoforms or post-translational modifications

• Subcellular localization inconsistencies:

  • Problem: Varying reports of nuclear envelope localization versus cytoplasmic distribution

  • Solution: Use fractionation methods combined with immunofluorescence; include co-staining with organelle markers; validate fixation methods as they can affect epitope accessibility

• Complex formation complications:

  • Problem: MTMR8-MTMR9 complex dissociates under serum starvation within 2 hours

  • Solution: Carefully control experimental conditions; consider crosslinking to stabilize complexes; monitor timing meticulously during serum deprivation experiments

• Optimization table for common applications:

By anticipating these common pitfalls and implementing the suggested solutions, researchers can significantly improve the reliability and reproducibility of their MTMR8 antibody-based experiments.

How can researchers effectively investigate the functional cooperation between MTMR8 and PI3K in actin filament modeling?

To comprehensively investigate the functional cooperation between MTMR8 and PI3K in actin filament modeling, researchers should implement a multi-faceted experimental approach:

• Combined inhibition studies:

  • Test MTMR8 knockdown alone (using morpholinos or siRNA)

  • Apply PI3K inhibitor LY294002 (10 µM) between 10-24 hpf in zebrafish or for appropriate intervals in cell culture

  • Examine combined effects of MTMR8 knockdown plus PI3K inhibition

  • Both MTMR8-MO1 and MTMR8-MO2+LY294002 lead to disorganization of the actin cytoskeleton, suggesting their functional cooperation

• PH/G domain function analysis:

  • Design constructs with PH/G domain deletions

  • Compare phenotypes of full-length versus PH/G-deleted MTMR8

  • While PH/G domain deletion alone may not produce obvious defects, it becomes critical when combined with PI3K inhibition

  • Monitor Akt phosphorylation levels as a readout of PI3K pathway activity (increased pAkt is observed in MTMR8 morphants)

• Actin cytoskeleton visualization techniques:

  • Employ phalloidin staining to visualize F-actin organization

  • Use time-lapse microscopy to monitor dynamic changes in actin filament modeling

  • Quantify actin filament length, orientation, and density

  • In muscle development studies, combine with antibody F59 staining, which detects mostly slow myofibrils

• Cell transplantation experiments:

  • Perform cell transplantation to determine whether MTMR8 functions cell-autonomously or non-cell-autonomously

  • Previous research indicates MTMR8 works in a non-cell autonomous manner in actin modeling

  • Design donor-to-host transplantation schemes with different combinations of MTMR8 knockdown and control cells

This comprehensive approach allows researchers to decipher the precise mechanisms by which MTMR8 cooperates with PI3K to regulate actin filament modeling and muscle development, potentially revealing new therapeutic targets for muscle disorders.

What methodological approaches are most effective for studying MTMR8's involvement in the Hedgehog signaling pathway?

To effectively investigate MTMR8's involvement in the Hedgehog (Hh) signaling pathway, researchers should employ the following methodological approaches:

• Genetic interaction studies:

  • Combine MTMR8 knockdown with manipulation of key Hh pathway components

  • Co-inject MTMR8 morpholino with dominant-negative PKA (dnPKA) mRNA, as PKA is a negative regulator of Hh signaling

  • Assess rescue effects: dnPKA co-injection rescues the muscle defects in MTMR8 morphants, supporting Hh pathway involvement

• Muscle development analysis:

  • Label slow muscle using antibody F59 (detects mostly slow myofibrils)

  • Quantify myofibril length and organization in control versus MTMR8-depleted embryos

  • MTMR8 morphants show shorter, reduced fibrils compared to controls

  • Use anti-Prox1 (slow muscle nuclear marker) to count muscle pioneer and slow muscle cells

• Hh pathway component analysis:

  • Monitor expression of Hh target genes using qRT-PCR or in situ hybridization

  • Examine protein levels and phosphorylation status of Gli transcription factors

  • Track Smoothened localization to primary cilia

  • Measure Gli-responsive luciferase reporter activity in control versus MTMR8-depleted cells

• Experimental matrix for Hh pathway analysis:

By systematically applying these approaches, researchers can establish whether MTMR8 functions as an upstream regulator, downstream effector, or modulator of the Hh pathway, providing insights into its role in developmental processes and potential involvement in Hh-related disorders.

How can researchers integrate phosphoinositide profiling with functional studies to understand MTMR8's role in cellular processes?

To comprehensively understand MTMR8's role in cellular processes, researchers should integrate phosphoinositide profiling with functional studies using the following methodological framework:

• Comprehensive phosphoinositide profiling:

  • Mass spectrometry-based analysis: Quantify changes in PtdIns(3)P, PtdIns(3,5)P₂, and PtdIns(5)P levels in response to MTMR8 manipulation

  • Fluorescent probe-based detection: Use specific antibodies or protein domains (e.g., FYVE domains) that recognize PtdIns(3)P

  • Comparative analysis: Examine MTMR8 alone versus MTMR8+MTMR9 effects on phosphoinositide levels

  • Spatial distribution mapping: Create subcellular maps of phosphoinositide distribution using high-resolution microscopy

• Integrative experimental design:

• Mechanistic dissection approaches:

  • Phosphatase-dead mutants: Create catalytically inactive MTMR8 (C338S mutation in the active site) to separate enzymatic from scaffolding functions

  • Domain-specific mutants: Test PH/G domain deletion to understand its role in phosphoinositide binding and PI3K pathway interaction

  • Substrate-specific variants: Engineer MTMR8 variants with altered substrate specificity through mutation of substrate-binding residues

  • Temporal manipulation: Use inducible expression/degradation systems to examine acute versus chronic effects of MTMR8 activity

• Systems biology integration:

  • Correlate phosphoinositide changes with transcriptomic and proteomic alterations

  • Map MTMR8-dependent phosphoinositide changes to specific organelle functions

  • Model the relationship between phosphoinositide conversion and downstream functional effects

  • Apply mathematical modeling to predict threshold effects and compensatory mechanisms

This integrated approach enables researchers to establish causal relationships between MTMR8's enzymatic activity on specific phosphoinositide substrates and the resulting cellular phenotypes, providing a comprehensive understanding of its role in diverse processes including autophagy, apoptosis, and cytoskeletal organization.

What emerging technologies could advance our understanding of MTMR8 function in disease models?

Several emerging technologies hold promise for advancing our understanding of MTMR8 function in disease models:

• CRISPR-based technologies:

  • Base editing and prime editing: Create precise mutations in MTMR8 to model disease-associated variants

  • CRISPRi/CRISPRa systems: Enable temporal and spatial control of MTMR8 expression

  • CRISPR screens: Identify synthetic lethal interactions and pathway components that modulate MTMR8 function

  • In vivo CRISPR editing: Generate tissue-specific MTMR8 knockout animal models to study its role in disease development

• Advanced imaging approaches:

  • Super-resolution microscopy: Track MTMR8 and phosphoinositide dynamics with nanometer precision

  • Live-cell phosphoinositide biosensors: Monitor real-time changes in PtdIns(3)P and PtdIns(3,5)P₂ pools

  • Correlative light and electron microscopy (CLEM): Examine MTMR8's role in membrane remodeling events during autophagy

  • Lattice light-sheet microscopy: Capture rapid phosphoinositide conversion events with minimal phototoxicity

• Structural biology innovations:

  • Cryo-EM analysis: Resolve the structure of MTMR8-MTMR9 complexes to understand activation mechanisms

  • AlphaFold-based modeling: Predict interaction interfaces between MTMR8 and binding partners

  • Hydrogen-deuterium exchange mass spectrometry: Map conformational changes upon complex formation

• Organoid and patient-derived models:

  • Human iPSC-derived organoids: Study MTMR8 function in physiologically relevant 3D tissue contexts

  • Patient-derived cells: Examine MTMR8 pathway dysregulation in cells from individuals with neuromuscular disorders

  • Tissue-on-chip platforms: Assess MTMR8's role in muscle development and disease in microfluidic devices

These technologies, when applied to MTMR8 research, could reveal new insights into its role in neuromuscular disorders, cancer, and other diseases where phosphoinositide signaling and membrane trafficking are dysregulated.

How might researchers investigate the potential therapeutic applications of modulating MTMR8 activity?

To investigate potential therapeutic applications of modulating MTMR8 activity, researchers should implement a comprehensive, translational research strategy:

• Target validation approaches:

  • Expression profiling: Analyze MTMR8 expression across disease tissues versus healthy controls

  • Genetic association studies: Identify MTMR8 variants associated with disease susceptibility or progression

  • Functional genomics: Employ CRISPR screens to determine contexts where MTMR8 modulation affects disease-relevant phenotypes

  • Conditional knockout models: Generate tissue-specific MTMR8 knockout animals to evaluate effects on disease development and progression

• Therapeutic modulation strategies:

• Preclinical validation methodologies:

  • Zebrafish models: Leverage established zebrafish systems to evaluate MTMR8 modulators in muscle development

  • Cell-based phenotypic screens: Assess effects on autophagy, actin organization, and cell survival

  • Ex vivo tissue assays: Test compounds on patient-derived muscle biopsies

  • Phosphoinositide profiling: Quantify restoration of normal phosphoinositide levels after treatment

• Translational considerations:

  • Biomarker development: Identify phosphoinositide signatures or downstream pathway readouts to monitor treatment efficacy

  • Delivery strategies: Develop tissue-specific delivery methods for MTMR8 modulators

  • Combination approaches: Test MTMR8 modulators with PI3K inhibitors or Hedgehog pathway modulators based on established functional connections

This systematic approach would enable researchers to translate fundamental insights about MTMR8's biochemical functions into potential therapeutic strategies for diseases associated with phosphoinositide dysregulation, autophagy defects, or muscle development abnormalities.

What is the most reliable protocol for validating a new MTMR8 antibody for research use?

A comprehensive validation protocol for new MTMR8 antibodies should include the following sequential steps:

• Initial specificity testing:

  • Western blot analysis: Test antibody against lysates from multiple cell lines known to express MTMR8

    • Recommended positive controls: HeLa cells, A375 cells, human placenta tissue

    • Expected molecular weight: 79 kDa (calculated), though 55-58 kDa is often observed

  • Knockdown validation: Compare signal in control versus MTMR8 siRNA-treated cells

    • Target knockdown efficiency: MTMR8 level <0.3 relative to control siRNA

    • Include appropriate loading controls (β-actin, GAPDH)

• Cross-reactivity assessment:

  • Overexpression system: Test antibody against cells overexpressing MTMR8 versus related family members (MTMR6, MTMR7)

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm signal specificity

  • Immunoprecipitation-mass spectrometry: Identify all proteins pulled down by the antibody

• Application-specific validation matrix:

• Lot-to-lot consistency verification:

  • Reference sample testing: Maintain validated reference samples to test each new antibody lot

  • Quantitative comparison: Use densitometry to compare signal intensity and specificity

  • Documentation: Maintain detailed records of validation results for each lot

• Functional validation:

  • MTMR9 co-immunoprecipitation: Verify ability to detect MTMR8-MTMR9 complexes

  • Phosphoinositide level monitoring: Confirm that antibody can detect changes in MTMR8 that correlate with altered phosphoinositide levels

By following this comprehensive validation protocol, researchers can ensure that their MTMR8 antibody provides reliable, specific detection across multiple applications, enhancing experimental reproducibility and data quality.

What are the optimal methodologies for studying MTMR8's enzymatic activity in vitro and in cells?

To effectively study MTMR8's enzymatic activity, researchers should employ complementary in vitro and cellular methodologies:

• In vitro phosphatase activity assays:

  • Radiolabeled substrate approach: Measure release of [³²P]-PO₄ from radiolabeled PtdIns(3)P and PtdIns(3,5)P₂ substrates

    • Assay both MTMR8 alone and MTMR8-MTMR9 complex (MTMR9 increases MTMR8 activity 4-fold toward PtdIns(3)P)

  • Malachite green phosphate detection: Colorimetric quantification of released phosphate

  • Fluorescent substrate assays: Use fluorescent phosphoinositide analogs for real-time activity monitoring

  • Enzyme kinetics determination: Measure K​m and V​max values toward different substrates

• Cellular phosphoinositide quantification:

  • Mass spectrometry-based lipidomics: Absolute quantification of phosphoinositide species

  • Immunofluorescence with anti-PtdIns(3)P antibodies: Count spots larger than 1 nm as individual PI(3)P molecules

    • Examine at least 50 cells per condition with three replicates

  • PtdIns(5)P production measurements: Quantify as a readout of PtdIns(3,5)P₂ dephosphorylation

  • Phosphoinositide biosensors: Express fluorescent protein-tagged lipid-binding domains that specifically recognize PtdIns(3)P

• Experimental design considerations:

• Advanced approaches:

  • Phosphatase-dead mutants: Create catalytically inactive MTMR8 (C338S mutation in active site)

  • Real-time activity monitoring: Use FRET-based biosensors to track phosphoinositide conversion events

  • Subcellular fractionation: Determine compartment-specific enzymatic activity

  • Conditional activation systems: Employ chemically-inducible dimerization to rapidly recruit MTMR8 to specific membranes