mtfr1l Antibody

What is MTFR1L and what cellular functions does it regulate?

MTFR1L (also known as FAM54B, HYST1888, MST116, MSTP116) is a mitochondrial protein that functions primarily as an anti-fusion regulator, negatively controlling mitochondrial fusion events by regulating OPA1 levels . Unlike what its name might suggest, MTFR1L does not directly promote mitochondrial fission but rather inhibits fusion processes.

MTFR1L is localized to the outer mitochondrial membrane (OMM) and is not inserted into the membrane but rather associated with its cytosolic face . It is ubiquitously expressed across tissues, with particularly high expression in brain, uterus, and heart . Recent research has identified MTFR1L as an AMPK substrate, with phosphorylation at two key residues (Ser103 and Ser238) that are critical for its function in controlling mitochondrial dynamics .

How should researchers select appropriate MTFR1L antibodies for their experiments?

When selecting MTFR1L antibodies, researchers should consider:

• Target species reactivity: Available antibodies show reactivity with human, mouse, and rat samples . Always verify cross-reactivity with your model organism.

• Validated applications: Different antibodies are validated for specific applications as shown in this comparison:

• Antigen region: Different antibodies target different regions of MTFR1L. For instance, PA555516 targets a sequence with high identity to mouse (96%) and rat (99%) orthologs , while PA555512 targets a sequence with 94% identity to mouse and 93% to rat orthologs .

What are the recommended protocols for detecting MTFR1L using Western Blot?

Based on the provided information for MTFR1L antibody applications in Western Blot:

• Recommended dilution: Use 1:500-1:2000 dilution for Western Blot applications .

• Expected molecular weight: MTFR1L is observed at approximately 37 kDa .

• Sample preparation: When working with MTFR1L, be aware that you may observe multiple bands in some preparations. The major functional isoform appears at ~38 kDa, while in some preparations a 25 kDa band may be detected that could represent a degradation product .

• Buffer conditions: For optimal results, store antibodies at -20°C in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Controls: For validation purposes, siRNA treatment against MTFR1L can serve as a negative control, showing a significant reduction of signal at mitochondria in immunofluorescence and reduced band intensity in Western Blot .

What methodologies are effective for studying MTFR1L subcellular localization?

Several experimental approaches have proven effective for determining MTFR1L's submitochondrial localization:

• Immunofluorescence co-localization: MTFR1L shows endogenous co-localization with the OMM marker TOM20, confirming its mitochondrial localization .

• Proteinase K degradation assay: Treatment of isolated mitochondria with increasing concentrations of proteinase K can reveal submitochondrial localization. MTFR1L degrades with similar patterns to OMM proteins like Mfn2 (at 1-5 μg/ml proteinase K), while intermembrane space and inner membrane proteins require higher concentrations (10-20 μg/ml) for degradation .

• Sodium carbonate extraction: Treatment of isolated mitochondria with sodium carbonate (pH 11) followed by centrifugation can determine membrane insertion status. MTFR1L assorts with non-inserted proteins like cytochrome c in the supernatant, indicating it is a labile protein associated with the cytosolic face of the OMM rather than being membrane-inserted .

How can researchers investigate AMPK-dependent phosphorylation of MTFR1L?

MTFR1L is phosphorylated by AMPK at Ser103 and Ser238, which is critical for its function. To study this:

• Phospho-specific antibodies: Use phospho-specific antibodies raised against MTFR1L pSer103 and pSer238 to detect phosphorylation status .

• Phosphorylation assays: Monitor MTFR1L phosphorylation under basal conditions and following AMPK activation with A-769662 (an AMPK activator). Expect increased phosphorylation intensity upon AMPK activation .

• Mutational analysis: Generate MTFR1L nonphosphorylatable mutant constructs (S103A and S238A) as negative controls for phosphorylation. Similarly, create phosphomimetic mutants (S103D/S238D) to study functional effects of constitutive "phosphorylation" .

• Validation in AMPK-α1α2 double KO cells: Use CRISPR-Cas9 to generate AMPK-α1α2 double knockout cells as a system to confirm AMPK-dependency of MTFR1L phosphorylation .

How can researchers investigate the role of MTFR1L in mitochondrial fusion and fission dynamics?

To study MTFR1L's role in mitochondrial dynamics, researchers can employ several approaches:

• MTFR1L knockout/knockdown experiments: Generation of MTFR1L KO cells or siRNA-mediated silencing induces mitochondrial hyperfusion, characterized by elongated mitochondria .

• Rescue experiments: Express wild-type MTFR1L or mutants in knockout cells to determine structural requirements for function. The N-terminal domain is required for both mitochondrial localization and morphology regulation .

• Double knockdown experiments: Silence both MTFR1L and OPA1 (a pro-fusion protein) to demonstrate the OPA1-dependency of the hyperfusion phenotype observed in MTFR1L KO cells .

• Electron microscopy analysis: Transmission electron microscopy (TEM) can reveal ultrastructural changes in mitochondrial cristae. MTFR1L KO cells exhibit increased cristae tightness characterized by decreased cristae width, similar to OPA1 overexpression .

• Apoptosis resistance assays: MTFR1L KO cells show resistance to cell death induced by ABT 737 and actinomycin D, with decreased levels of cleaved caspase 3 and reduced cytosolic cytochrome c .

What are the critical technical considerations when using MTFR1L antibodies for immunoprecipitation?

When performing immunoprecipitation (IP) with MTFR1L antibodies:

• Recommended antibody amount: Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Verified sample types: Successful IP has been demonstrated with mouse testis tissue .

• Titration requirements: It is recommended to titrate the antibody in each testing system to obtain optimal results, as the ideal concentration can be sample-dependent .

• Buffer conditions: For long-term storage of the antibody, store at -20°C and avoid freeze/thaw cycles to maintain activity .

How can researchers assess mitochondrial function in relation to MTFR1L activity?

When investigating functional consequences of MTFR1L manipulation:

• Respirometry: Use high-resolution respirometry to measure oxygen consumption in intact cells. Parameters to analyze include:

  • Basal (ROUTINE) respiration

  • Non-phosphorylating respiration (LEAK) after addition of oligomycin

  • Maximal electron transport system capacity (ETS) after CCCP titration

  • Residual oxygen consumption after addition of antimycin A and rotenone

• ATP/ADP ratio measurement: Use luciferin bioluminescence to monitor changes in ATP/ADP ratio under steady state or during glucose starvation .

• Response to ETC inhibitors: Treatment with respiratory complex inhibitors (rotenone, antimycin A) can reveal differential responses in MTFR1L KO vs. wild-type cells. MTFR1L KO cells show decreased AMPK activation in response to these inhibitors, suggesting that mitochondrial hyperfusion may protect from ETC-induced dysfunction .

• AMPK activation monitoring: Track phosphorylation of AMPK and its substrate ACC as a readout of cellular energetic stress in relation to MTFR1L function .

How does MTFR1L interact with other members of the MTFR family, and what unique functions does it serve?

• Evolutionary divergence: Investigate whether MTFR1, MTFR2, and MTFR1L have diverged during evolution and acquired specialized roles in controlling mitochondrial dynamics in response to different stimuli .

• Mechanism comparison: Determine whether other MTFR1 family members (MTFR1/MTFR2) regulate mitochondrial morphology through similar mechanisms as MTFR1L (OPA1 regulation) or by directly controlling the mitochondrial fission process .

• Tissue-specific functions: Given the differential expression pattern of MTFR1L across tissues (enriched in brain, uterus, and heart), explore potential tissue-specific functions or regulation mechanisms .

What experimental approaches can be used to study MTFR1L function in neuronal systems?

MTFR1L has been studied in neuronal systems, with implications for dendritic mitochondrial distribution. Researchers can:

• In utero electroporation (IUE): Perform CA1-targeted IUE with combinations of:

  • Cytoplasmic fluorescent proteins (tdTomato, mGreenLantern)

  • Mitochondrial matrix-targeted fluorescent proteins (mt-YFP, mt-DsRed)

  • Control non-targeting shRNA (shNT) or shRNA targeting MTFR1L

• Compartment-specific analysis: Analyze mitochondrial morphology in different dendritic compartments:

  • Basal (stratum oriens, SO)

  • Apical oblique (stratum radiatum, SR)

  • Apical tuft (stratum lacunosum-moleculare, SLM)

• Rescue experiments: Co-express MTFR1L-targeting shRNA with shRNA-resistant human cDNA expressing hMTFR1L to confirm specificity .

• Functional rescue: Test the hypothesis that mitochondrial elongation mediated by MTFR1L knockdown can be rescued by simultaneously knocking down Opa1 in CA1 pyramidal neurons .