mtfr2 Antibody

What is MTFR2 and why is it important for cellular function?

MTFR2 (also known as DUFD1 or FAM54A) is a mitochondrial outer membrane protein that plays a crucial role in DRP1-dependent mitochondrial fission. Research has demonstrated that MTFR2 is co-transcribed with core centromere/kinetochore components, suggesting its involvement in mitosis regulation. MTFR2 is particularly important for ensuring proper spindle integrity and chromosomal stability during cell division. Knockout of MTFR2 leads to prolonged mitotic duration, increased chromosome mis-segregation, and the formation of multi-nucleated daughter cells, highlighting its essential role in maintaining genomic stability .

What are the optimal fixation methods for MTFR2 antibody in immunofluorescence?

When performing immunofluorescence with MTFR2 antibodies, standard PFA (paraformaldehyde) fixation protocols are generally effective. Based on published methodologies, cells are typically fixed with 4% PFA for approximately 15 minutes at room temperature. For mitochondrial co-localization studies, it's recommended to use Mito-Tracker Red for mitochondrial labeling before fixation. As demonstrated in research protocols, MTFR2 can be detected using rabbit anti-MTFR2 antibody followed by Alexa Fluor 488 conjugated secondary antibody, while DNA can be counterstained with DAPI for nuclear visualization .

How can I validate the specificity of my MTFR2 antibody?

Validation of MTFR2 antibody specificity should involve multiple complementary approaches:

• Knockout/knockdown controls: Compare staining between wild-type cells and MTFR2 knockout or knockdown cells to confirm signal specificity.

• Overexpression validation: Transfect cells with MTFR2-GFP and demonstrate co-localization with the antibody signal.

• Western blot analysis: Verify that the antibody detects bands of appropriate molecular weight (both endogenous MTFR2 and MTFR2-GFP in transfected cells).

• Subcellular localization: Confirm that the detected signal colocalizes with mitochondrial markers like TOM20 or Mito-Tracker.

Published research has validated MTFR2 antibodies by demonstrating their specificity in detecting endogenous MTFR2 alongside MTFR2-GFP of expected sizes in immunoblot analyses .

What are the recommended protocols for using MTFR2 antibody in Western blotting?

For optimal Western blot detection of MTFR2, the following protocol has been successfully implemented in research studies:

• Sample preparation: Lyse cells with RIPA buffer for 30 minutes on ice, followed by centrifugation at 12,000g for 15 minutes at 4°C.

• Protein quantification: Determine protein concentration using a BCA protein assay kit.

• Antibody dilution: Use anti-MTFR2 antibody at a 1:500 dilution (Sigma-Aldrich, HPA029792).

• Controls: Include GAPDH (1:1,000, Abcam, ab181602) as a loading control.

• Detection: Utilize ECL Chemiluminescent Substrate Reagent Kits for visualization.

This protocol has successfully detected both endogenous MTFR2 and exogenous MTFR2-GFP in experimental settings .

How should I design experiments to study MTFR2 phosphorylation during mitosis?

Investigating MTFR2 phosphorylation during mitosis requires careful experimental design:

• Cell synchronization: Utilize cell cycle synchronization methods such as:

  • Thymidine (2mM) for 24 hours to arrest cells at G1/S boundary

  • RO3306 (10μM) for at least 6 hours to arrest cells at G2/M boundary

  • Nocodazole (330nM) to arrest cells at prometaphase

  • MG132 (10μM) to inhibit anaphase onset

• Phosphorylation analysis:

  • Perform immunoblotting with phospho-specific antibodies if available

  • Use phosphatase inhibitors in lysis buffers

  • Consider Phos-tag gels to separate phosphorylated from non-phosphorylated forms

  • Analyze phosphorylation site mutations (like the MTFR2-11A mutant where 11 serine/threonine sites are mutated)

• PLK1 inhibition: Consider using PLK1 inhibitors like BI2536 to study the relationship between PLK1 activity and MTFR2 phosphorylation .

What controls should be included when studying MTFR2 knockdown/knockout effects?

When investigating the effects of MTFR2 depletion, include these essential controls:

• Rescue experiments: Transfect MTFR2 knockout cells with wild-type MTFR2 to confirm that observed phenotypes are directly attributable to MTFR2 loss.

• Multiple knockout/knockdown strategies: Utilize both siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout to rule out off-target effects.

• Time-course analysis: For inducible systems, perform time-course experiments to track the progression of phenotypes following MTFR2 depletion.

• Cancer-associated variants: Include cancer-associated MTFR2 mutants (such as E126Q from renal clear cell carcinoma or R290Q from colorectal cancer) as functional controls.

• Mitochondrial dynamics controls: Include other mitochondrial fission/fusion protein manipulations (like MFN1 overexpression or DRP1-K38A expression) to contextualize MTFR2-specific effects versus general mitochondrial dynamics disruption .

How does MTFR2 expression correlate with cancer progression and patient outcomes?

Multiple studies have established significant correlations between MTFR2 expression and cancer:

Breast Cancer:

• 607 out of 1000 breast cancer patients showed high MTFR2 expression

• MTFR2 expression was significantly higher in breast cancer tissues compared to adjacent normal tissues (p=0.016)

• High MTFR2 expression correlated with:

  • Patient age (p=0.001)

  • Tumor grade (p=0.009)

  • Lymph node metastasis (p=0.010)

  • HER2 status (p=0.016)

• Multivariate analysis identified MTFR2 expression as an independent prognostic factor (HR: 1.96, 95% CI: 1.55-2.48, p=0.03)

Glioma:

• MTFR2 was significantly elevated in glioma samples

• Higher MTFR2 expression correlated with poor prognosis

These findings consistently demonstrate that MTFR2 overexpression is associated with more aggressive disease and worse clinical outcomes across multiple cancer types.

What methods can be used to study the relationship between MTFR2 expression and immune cell infiltration in tumors?

To investigate associations between MTFR2 and tumor immune microenvironment:

• Single-sample Gene Set Enrichment Analysis (ssGSEA):

  • Utilize the GSVA package via R software

  • Calculate levels of 24 tumor-infiltrated immune cell types

  • Determine correlation between MTFR2 expression and immune cell infiltration using Spearman correlation

  • Apply Wilcoxon rank-sum test to analyze the association between different MTFR2 expression levels and immune cell infiltration

• Immunohistochemistry validation:

  • Perform IHC on tissue microarrays containing tumor samples

  • Score MTFR2 expression levels

  • Co-stain with immune cell markers

  • Analyze correlation between MTFR2 expression and immune cell presence

• Gene Set Enrichment Analysis (GSEA):

  • Utilize R package and clusterProfiler to identify differential pathways between high and low MTFR2 expression groups

  • Perform gene set permutations (1,000 times recommended for each analysis)

  • Adopt pathways with adjusted p-value <0.05, FDR q-value <0.25, and NES >1.0 as significantly enriched

How can I effectively analyze MTFR2 mutations found in cancer samples?

When analyzing cancer-associated MTFR2 mutations:

• Data retrieval: Access MTFR2 sequence variants from cancer databases such as:

  • cBIOPORTAL

  • TumorPortal

  • COSMIC

• Mutation classification:

  • Focus on truncations caused by nonsense or splicing mutations

  • Identify recurring mutants at the same residue across multiple samples

  • Use Mutation Assessor to evaluate mutation impact (prioritize medium to high impact mutations)

• Functional validation: Introduce mutations into MTFR2 expression constructs and assess their impact on:

  • Mitochondrial fission capability

  • Mitotic progression

  • Spindle integrity

  • Chromosome segregation

Existing research has identified several cancer-associated MTFR2 variants that fail to induce mitochondrial fragmentation, with exceptions like E126Q (renal clear cell carcinoma) and R290Q (colorectal cancer) that retain partial function .

How does MTFR2 influence mitochondrial dynamics during cell division?

MTFR2 plays a critical role in coordinating mitochondrial fission during mitosis:

• Mitotic mitochondrial fragmentation: MTFR2 promotes DRP1-dependent mitochondrial fission, which is essential during mitosis. This fragmentation ensures proper mitochondrial segregation to daughter cells.

• Impact on spindle integrity: MTFR2 knockout leads to two major spindle defects:

  • Approximately 32.5% of cells harbor multi-polar spindles (compared to 7.5% in control cells)

  • Around 47.5% lack astral microtubules (compared to 20.0% in control cells)

  • Cells lacking astral microtubules tend to have shorter spindles

• Centrosome effects: MTFR2 knockout cells show:

  • Lower γ-Tubulin intensity at spindle poles, indicating reduced microtubule nucleating activities

  • Centriole splitting or loss, with some spindle poles having none or only one CENTRIN-1 dot associating with γ-Tubulin foci

These findings indicate that MTFR2-mediated mitochondrial fragmentation during mitosis is essential for maintaining spindle integrity and ensuring proper chromosome segregation.

What experimental approaches can reveal the mechanisms of MTFR2's impact on spindle integrity?

To investigate how MTFR2 affects spindle integrity:

• Live cell imaging:

  • Transfect cells with mCherry-Tubulin to visualize spindle dynamics

  • Compare control and MTFR2 knockout cells throughout mitosis

  • Measure spindle oscillation and pole-to-cortex distances

  • Track chromosome segregation errors

• Immunofluorescence analysis:

  • Stain for spindle components (β-Tubulin), centrosome markers (γ-Tubulin, CENTRIN-1), and mitochondria (TOM20)

  • Quantify spindle pole abnormalities, spindle length, and astral microtubule presence

  • Assess cold-stable kinetochore microtubules to evaluate attachment stability

• Functional rescue experiments:

  • Express wild-type MTFR2 vs. phosphorylation mutants (MTFR2-11A) in knockout cells

  • Compare ability to rescue spindle defects

  • Test cancer-associated variants to assess their impact on spindle integrity

How do MTFR2 and DRP1 interact in mitochondrial fission regulation?

The relationship between MTFR2 and DRP1 in mitochondrial fission involves:

• DRP1 recruitment: While MTFR2 participates in DRP1-dependent mitochondrial fission, knockout of MTFR2 does not prevent DRP1 recruitment to mitochondria. Immunofluorescence studies show that:

  • DRP1 signals overlap with mitochondria (defined by TOM20 signals) similarly in control and MTFR2 knockout cells

  • Mander's overlap coefficients show no significant difference in DRP1-mitochondria association

• Protein interaction analysis:

  • Direct interactions between MTFR2 and DRP1 were not detected in GFP-trap experiments using cell lysates from MTFR2-GFP expressing cells

  • This suggests MTFR2 may influence DRP1 function without direct binding

• Subcellular fractionation:

  • Analysis of whole cell lysates, mitochondrial fractions, and cytosolic fractions showed similar DRP1 distribution in control and MTFR2 knockout cells

  • This further supports that MTFR2 does not affect DRP1 recruitment to mitochondria

What are the key phosphorylation sites in MTFR2 and how do they affect its function?

MTFR2 phosphorylation is a critical regulatory mechanism:

• Key phosphorylation sites: Research has identified 11 important serine/threonine phosphorylation sites in MTFR2:

  • S119, S291, S305, S311, S314, S328, S335, S342, S354, T378, and S379

• Functional significance:

  • MTFR2 is phosphorylated during mitosis

  • Phosphorylation mutants (MTFR2-11A, where all 11 sites are mutated) fail to correct the prolonged mitotic duration seen in MTFR2 knockout cells

  • This indicates phosphorylation is essential for MTFR2's mitotic functions

• Relation to cancer variants:

  • Cancer-associated MTFR2 variants also failed to correct prolonged mitotic duration

  • This suggests that both phosphorylation and cancer mutations affect similar functional aspects of MTFR2

What are common challenges when using MTFR2 antibodies for immunofluorescence and how can they be addressed?

Researchers frequently encounter these challenges when using MTFR2 antibodies:

• High background signal:

  • Increase blocking time (use 5% BSA or normal serum for 1-2 hours)

  • Optimize antibody dilution (typically 1:200-1:500 range)

  • Include additional washing steps (5-6 washes of 5 minutes each)

  • Pre-absorb secondary antibodies with cell/tissue lysates

• Weak mitochondrial signal:

  • Verify mitochondrial marker co-localization (Mito-Tracker Red or TOM20 antibody)

  • Optimize fixation method (test both methanol and PFA fixation)

  • Use antigen retrieval methods if necessary

  • Ensure cells aren't overly confluent which can affect mitochondrial morphology

• Variability between experiments:

  • Standardize cell culture conditions (passage number, confluency)

  • Include positive controls (MTFR2-GFP transfected cells)

  • Maintain consistent image acquisition settings

  • Perform parallel staining of control and experimental samples

How can I quantitatively assess mitochondrial morphology changes in MTFR2 studies?

For rigorous quantification of mitochondrial morphology:

• Morphological classification system:

  • Categorize mitochondrial morphologies into distinct phenotypes:

    • Tubular: elongated, networked mitochondria

    • Fission: fragmented but not hyperfragmented

    • Hyper-fission: extensively fragmented mitochondria

  • Score at least 100 cells per condition across three independent experiments

• Quantitative measurement approaches:

  • Measure mitochondrial length in control and MTFR2 knockout/overexpressing cells

  • Calculate mitochondrial footprint area

  • Determine mitochondrial interconnectivity (ratio of area to perimeter)

  • Use automated image analysis software (ImageJ with mitochondrial analysis plugins)

• Complementary functional assays:

  • Measure mitochondrial membrane potential

  • Assess mitochondrial respiration

  • Evaluate mitochondrial ROS production

  • Perform mitochondrial mobility tracking