MTMR4 Antibody

What cellular processes is MTMR4 involved in, and why is antibody detection important?

MTMR4 has been established as a key player in several critical cellular pathways:

• Endosomal trafficking and signaling: MTMR4 resides primarily in early endosomes through its FYVE domain and regulates the duration of PtdIns(3)P signaling

• Signaling pathway modulation: MTMR4 functions as a negative regulator of both TGFβ and BMP signaling pathways by dephosphorylating activated R-Smads

• Phagocytic processes: MTMR4 negatively regulates FcγR surface expression and phagocytosis in macrophages

• Autophagy regulation: MTMR4 is essential for proper autophagosome and autolysosome formation

Antibody detection enables precise tracking of MTMR4's subcellular localization and dynamic recruitment during these processes, particularly during signaling events and membrane trafficking.

What are the key considerations when selecting an MTMR4 antibody for research applications?

When selecting an MTMR4 antibody, researchers should consider:

• Verified reactivity: Confirm the antibody's reactivity with your species of interest (human, mouse, rat)

• Validated applications: Ensure the antibody is validated for your specific application (WB, IP, IF, IHC, ELISA)

• Recognition domain: Some antibodies target specific domains of MTMR4; for instance, antibodies targeting AA 1155-1187 recognize the C-terminal region

• Observed molecular weight: While the calculated molecular weight of MTMR4 is 133 kDa, it typically appears at 160-170 kDa on Western blots due to post-translational modifications

How can MTMR4 antibodies be used to study its role in attenuating TGFβ and BMP signaling pathways?

MTMR4 acts as a negative regulator of both TGFβ and BMP signaling pathways. To study this:

• Co-immunoprecipitation experiments:

  • Use MTMR4 antibodies to immunoprecipitate endogenous MTMR4 from cells before and after TGFβ or BMP stimulation

  • Probe for R-Smads (Smad1, Smad2, Smad3) in the immunoprecipitate to detect interaction

  • The interaction between MTMR4 and R-Smads is transient and peaks after TGFβ stimulation, decreasing as phosphorylation levels decline

• Combined immunofluorescence and phospho-specific antibodies:

  • Treat cells with TGFβ or BMP ligands at various time points

  • Use MTMR4 antibodies alongside phospho-Smad antibodies to track:

    • Co-localization in early endosomes

    • Dephosphorylation kinetics of activated Smads

    • Nuclear translocation of Smads

• MTMR4 knockdown/overexpression coupled with reporter assays:

  • Establish cells with MTMR4 knockdown or overexpression

  • Measure pathway activity using pathway-specific luciferase reporters (3TP-lux, CAGA-lux for TGFβ; BRE-lux and GCCG-lux for BMP)

  • Validate findings using Western blotting with MTMR4 antibodies to confirm expression levels

What methodological approaches can resolve contradictory results in MTMR4 studies?

When facing contradictory results in MTMR4 studies, consider these methodological approaches:

• Domain-specific functional analysis:

  • The FYVE domain is critical for MTMR4 localization to early endosomes

  • MTMR4ΔFYVE1 shows diffused cytoplasmic distribution and reduced ability to dephosphorylate Smad3

  • The DSP (dual-specificity phosphatase) domain is essential for MTMR4's phosphatase activity

  • The C407S mutation abolishes phosphatase activity without affecting localization

  Testing constructs with specific domain mutations can clarify discrepancies between studies.

• Cell-type specific differences:

  • Use multiple cell lines to validate findings (e.g., PAE, HaCaT, L17, RAW 264.7)

  • Verify endogenous MTMR4 expression levels across cell types using Western blotting

  • Ensure phenotypes are rescued by re-introduction of siRNA-resistant MTMR4

• Temporal dynamics consideration:

  • MTMR4 exhibits dynamic recruitment to cellular structures during processes like phagocytosis

  • Design time-course experiments with multiple sampling points to capture transient interactions

  • Use live-cell imaging with fluorescently tagged MTMR4 to track these dynamics

How can MTMR4 antibodies help elucidate its role in macrophage phagocytosis?

MTMR4 has been shown to negatively regulate FcγR-mediated phagocytosis in macrophages. To study this:

• Quantitative immunofluorescence approach:

  • Use MTMR4 antibodies to track recruitment to forming phagosomes during FcγR-mediated phagocytosis

  • Combine with F-actin staining to quantify actin assembly at phagocytic cups

  • In MTMR4-overexpressing cells, a 20% reduction in F-actin intensity at phagocytic cups is observed

  • In MTMR4-knockdown cells, a 57% increase in F-actin intensity is observed

• FcγR surface expression analysis:

  • Use flow cytometry to measure FcγR surface levels in conjunction with MTMR4 manipulation

  • MTMR4 overexpression decreases FcγRI surface expression by approximately 20%

  • MTMR4 knockdown increases both FcγRI and FcγRII/III surface expression

• Phagosomal maturation studies:

  • Track PtdIns(3)P duration on phagosomes using PtdIns(3)P biosensors alongside MTMR4 antibody staining

  • MTMR4 knockdown increases PtdIns(3)P duration on phagosomes

  • Combine with bacterial infection models (e.g., Mycobacterium marinum) to study resistance to pathogen-induced phagosome arrest

What experimental controls are critical when studying MTMR4's effect on pathogen-containing phagosomes?

When investigating MTMR4's role in pathogen-containing phagosomes, these controls are essential:

• MTMR4 phosphatase-dead mutant controls:

  • Include the MTMR4-C407S mutant, which lacks phosphatase activity but maintains localization

  • This distinguishes between effects dependent on phosphatase activity versus protein-protein interactions

• Pathogen viability controls:

  • Use both viable and heat-killed bacteria to distinguish between active pathogen manipulation and passive processes

  • Compare pathogenic mycobacteria with non-pathogenic species or mutants lacking virulence factors

• Membrane trafficking markers:

  • Track multiple phagosomal maturation markers (Rab5, Rab7, LAMP1) alongside MTMR4

  • MTMR4 knockdown accelerates maturation of phagosomes containing pathogenic mycobacteria

• Mycobacterial phosphatase comparison:

  • Pathogenic mycobacteria secrete 3-phosphatases that degrade PtdIns(3)P to arrest phagosomal maturation

  • Compare MTMR4 knockdown effects with bacterial phosphatase inhibition to distinguish host versus pathogen effects

How can researchers accurately track MTMR4's role in autophagosome and endosome dynamics?

MTMR4 regulates the endocytic and autophagic pathways through its phosphatase activity. Advanced approaches include:

• Live-cell imaging with dual fluorescent markers:

  • Express fluorescently-tagged MTMR4 alongside markers for early endosomes (EEA1), late endosomes (Rab7), and autophagosomes (LC3)

  • MTMR4 localizes primarily to late endosomes and autophagosomes

  • MTMR4 knockdown results in:

    • Decreased motility, fusion, and fission of PI(3)P-enriched structures

    • Decreases in late endosomes, autophagosomes, and lysosomes

    • Enlargement of PI(3)P-enriched early and late endosomes

• Starvation-induced autophagy assays:

  • Subject cells to amino acid and serum starvation while monitoring MTMR4 localization

  • MTMR4 knockdown in starved cells shows:

    • Decreased autophagosomes and autolysosomes

    • Increased PI(3)P-containing autophagosomes and late endosomes

    • Impaired fusion of autophagosomes and late endosomes with lysosomes

• TFEB nuclear translocation analysis:

  • MTMR4 knockdown inhibits nuclear translocation of transcription factor-EB (TFEB) during starvation

  • This leads to reduced expression of lysosome-related genes

  • Track this using nuclear/cytoplasmic fractionation and immunoblotting with MTMR4 and TFEB antibodies

What innovative approaches can detect MTMR4's interaction with phosphoinositides in subcellular compartments?

To detect MTMR4's interaction with phosphoinositides in subcellular compartments:

• Proximity ligation assays (PLA):

  • Use MTMR4 antibodies together with antibodies against PtdIns(3)P or PtdIns(3,5)P₂

  • This technique detects proteins within 40 nm of each other, generating fluorescent puncta

  • Quantify PLA signals in various subcellular compartments using compartment-specific markers

• Phosphoinositide biosensors with MTMR4 manipulation:

  • Express fluorescent phosphoinositide biosensors (e.g., 2xFYVE domain for PtdIns(3)P)

  • Manipulate MTMR4 levels through knockdown or overexpression

  • MTMR4 overexpression decreases PtdIns(3)P duration on phagosomes

  • MTMR4 knockdown increases PtdIns(3)P duration and amplitude

• Phosphoinositide mass spectrometry:

  • Use lipidomic approaches to quantify absolute changes in phosphoinositide species

  • Compare phosphoinositide profiles in subcellular fractions from control versus MTMR4-manipulated cells

  • This provides direct quantification of MTMR4's enzymatic activity on endogenous substrates

What strategies help resolve inconsistent MTMR4 detection in Western blotting?

Researchers facing difficulties with MTMR4 detection should consider:

• Protein extraction optimization:

  • MTMR4 is membrane-associated through its FYVE domain

  • Include detergents suitable for membrane protein extraction (e.g., NP-40, Triton X-100)

  • Sonication may improve extraction from membrane fractions

• Molecular weight considerations:

  • While the calculated molecular weight is 133 kDa, MTMR4 typically appears at 160-170 kDa on Western blots

  • This discrepancy is likely due to post-translational modifications

  • Use positive control lysates from cells known to express MTMR4 (MCF-7, Jurkat, HepG2, HeLa cells)

• Antibody selection guidelines:

  • Different antibodies detect different epitopes with varying efficiency

  • For full-length MTMR4, antibodies targeting AA 1-1195 are recommended

  • For detecting C-terminal fragments, antibodies against AA 1155-1187 region are effective

• Sample preparation considerations:

  • Reduce heating time during sample preparation as MTMR4 is a large protein

  • Use lower percentage gels (6-8%) for better resolution of high molecular weight proteins

  • Consider using gradient gels (4-15%) to resolve potential MTMR4 degradation products

How can researchers validate contradictory findings regarding MTMR4's role in ion channel regulation and disease severity?

For researchers studying MTMR4's emerging role in disease contexts like Long QT Syndrome:

• Comprehensive SNV characterization:

  • MTMR4 SNVs have been associated with modulating ion channel degradation and clinical severity in long QT syndrome

  • Sequence MTMR4 in multiple cohorts with the same primary mutation but varying disease severity

  • Identify variants that consistently correlate with clinical phenotypes

• Cross-validation approaches:

  • Use multiple antibodies targeting different MTMR4 epitopes

  • Validate antibody specificity using MTMR4 knockout or knockdown controls

  • Perform parallel analyses with different methodologies (WB, IF, IP) to confirm consistency

• Functional validation in disease-relevant systems:

  • Use induced pluripotent stem cells (iPSCs) from patients with and without MTMR4 variants

  • Differentiate into disease-relevant cell types (e.g., cardiomyocytes for LQTS)

  • Confirm MTMR4 expression and localization with validated antibodies

  • Measure functional outcomes specific to the disease context (e.g., ion channel trafficking)

• Interactome analysis:

  • Perform immunoprecipitation with MTMR4 antibodies followed by mass spectrometry

  • Compare interactome profiles between normal and disease conditions

  • Identify disease-specific interaction partners (e.g., Nedd4L in LQTS)

  • Validate key interactions with co-immunoprecipitation and proximity ligation assays