Recombinant Bovine Transmembrane protein 11, mitochondrial (TMEM11)

What is the current understanding of TMEM11's subcellular localization?

TMEM11 was initially reported as an inner mitochondrial membrane protein, but more recent studies provide compelling evidence that it localizes to the outer mitochondrial membrane (OMM). This localization has been definitively demonstrated through multiple experimental approaches:

Methodological approach:

• Protease protection assays: When intact mitochondria were isolated from U2OS cells and treated with proteinase K, TMEM11 was completely digested similar to TOMM20 (an established OMM protein), while the inner membrane protein MIC60 remained protected unless the outer membrane was ruptured .

• APEX2 labeling: Electron microscopy visualization using APEX2-GFP-TMEM11 showed DAB precipitate on the exterior of mitochondria, consistent with OMM localization .

• Topology analysis: The N-terminal region of TMEM11 faces the cytosol, while the C-terminal region contains the transmembrane domain .

This subcellular localization correction is critical for properly interpreting TMEM11's protein interactions and functional roles.

How does TMEM11 affect mitochondrial morphology?

TMEM11 plays a significant role in maintaining normal mitochondrial morphology across different species.

Key experimental findings:

• In TMEM11-depleted U2OS cells, mitochondria become enlarged and bulbous compared to the narrow tubular mitochondria in control cells .

• Electron microscopy analysis reveals that TMEM11 depletion results in:

  • Enlarged mitochondria

  • Curved and/or highly elongated cristae membranes that frequently span the width of enlarged mitochondria

• Similar phenotypes were observed in Drosophila PMI (TMEM11 homolog) mutant flies, suggesting evolutionary conservation of this function .

Methodological approaches:

• CRISPR interference (CRISPRi) with sgRNAs targeting TMEM11

• Vital dye (Mitotracker) staining followed by fluorescence microscopy

• Transmission electron microscopy for ultrastructural analysis

What are the primary protein interaction partners of TMEM11?

TMEM11 interacts with multiple proteins that contribute to its diverse functions:

The interaction with BNIP3/BNIP3L appears particularly significant, as these proteins were the top scoring interactors in proteomic analysis . Direct binding between TMEM11 and these mitophagy receptors has been confirmed through yeast two-hybrid assays .

What are the methodological considerations for investigating TMEM11's role in mitophagy?

TMEM11 has been identified as a negative regulator of BNIP3/BNIP3L-dependent receptor-mediated mitophagy. When designing experiments to investigate this function, researchers should consider:

Methodological approaches:

• Mitophagy induction protocols:

  • Chemical inducers: CoCl₂ treatment (500 µM, 24h) to mimic hypoxia and upregulate BNIP3/BNIP3L expression

  • Physiological inducers: Actual hypoxia chambers for more physiologically relevant conditions

• Mitophagy quantification:

  • Mito-mKeima assay: This pH-sensitive fluorescent protein changes emission wavelength in acidic environments, allowing quantification of mitochondria in autolysosomes

  • Co-localization analysis: Immunofluorescence microscopy to detect mitophagy receptors (BNIP3/BNIP3L), autophagy markers (LC3), and mitochondrial markers

  • Western blot analysis of mitochondrial protein degradation

• Key controls and experimental design:

  • BNIP3/BNIP3L double knockdown to confirm mitophagy pathway specificity

  • Time course experiments (e.g., 24h, 48h post-induction)

  • Combined genetic manipulations (e.g., TMEM11 knockdown + BNIP3/BNIP3L knockdown)

Important findings to consider:

• TMEM11 depletion increases BNIP3/BNIP3L-dependent mitophagy in both normal and hypoxia-mimetic conditions

• TMEM11 co-enriches with BNIP3 and BNIP3L at discrete focal sites during mitophagy induction (65-70% co-enrichment)

• These focal sites are positive for LC3, indicating they are sites of mitophagosome formation

How should researchers approach the generation and validation of TMEM11 knockout mouse models?

Based on the literature, both conventional knockout and tissue-specific transgenic approaches have been successfully implemented for TMEM11. Key considerations include:

Generation strategies:

• CRISPR/Cas9-mediated knockout:

  • Target selection: Exon 2 of Tmem11-201 transcript contains most coding sequence and is recommended as the knockout region

  • sgRNA design: Both genome-wide off-target prediction and targeted validation should be performed

  • Genotyping primers:

    • F1: 5′-CTCCAATCTGAATACATCCAAAGTCC-3′

    • R1: 5′-GGCATTTCAGAATCTGGGTTAGTACAC-3′

    • F2: 5′-TGCTGTCTTACCTGTGAACTTGAGAGA-3′

    • R2: 5′-CTGTTTGTCAGGAGAAATGAGTGAGC-3′

• Tissue-specific overexpression (transgenic):

  • Promoter selection: α-myosin heavy chain (MHC) promoter for cardiac-specific expression

  • Vector construction: pαMHC-clone26 has been successfully used

Validation approaches:

• Protein expression: Western blotting to confirm complete protein loss

• Phenotypic analysis: Mitochondrial morphology assessment (critical for confirming functional impact)

• Subcellular localization studies: In wild-type controls, TMEM11 localizes to nucleus, cytoplasm, and mitochondria in cardiomyocytes

Functional characterization:

• For cardiac studies: Echocardiography, cardiac injury models (MI), cardiomyocyte proliferation assays

• For immune cells: Macrophage polarization, inflammasome activation, cytokine production

What experimental approaches can resolve apparent contradictions in TMEM11 research findings?

Several apparent contradictions exist in the TMEM11 literature that researchers should address:

Subcellular localization contradiction:

• Early studies: Reported TMEM11 as inner mitochondrial membrane protein

• Recent studies: Demonstrated outer mitochondrial membrane localization

• Resolution approaches:

  • Multi-method localization determination: Combine protease protection assays, APEX2 labeling, and biochemical fractionation

  • Domain-specific antibodies or tags to determine topology

  • Cross-species comparisons to determine if localization differences might be species-specific

Effect on MICOS complex contradiction:

• Some studies: Suggest TMEM11 impacts MICOS complex

• Other studies: Show TMEM11 stability is unaffected by MIC60 depletion, and TMEM11 depletion does not affect MICOS assembly

• Resolution approaches:

  • 2D BN-PAGE analysis of MICOS complex components in the presence/absence of TMEM11

  • Proximity labeling techniques (BioID, APEX) to map spatial relationships

  • Crosslinking mass spectrometry to identify direct protein interactions

Cellular function contradictions:

• Drosophila studies: Show significant metabolic impacts of PMI/TMEM11 loss

• Mammalian studies: Report varied phenotypes across different cell types

• Resolution approaches:

  • Cell type-specific conditional knockouts

  • Cross-species rescue experiments

  • Careful phenotypic comparison with standardized assays

What are the current methodological approaches for studying TMEM11's role in cardiomyocyte proliferation?

Recent research has uncovered a novel function of TMEM11 in regulating cardiomyocyte proliferation through RNA methylation. Key methodological considerations include:

Experimental models:

• In vitro approaches:

  • Primary neonatal cardiomyocyte cultures

  • Cardiomyocyte proliferation assays: EdU incorporation, Ki67 staining, pH3 staining[3,

• In vivo approaches:

  • TMEM11 knockout and cardiac-specific transgenic mice

  • Myocardial infarction models to assess regenerative capacity

  • Echocardiography for functional assessment

Molecular pathway analysis:

• m7G RNA methylation:

  • RNA methylation analysis to detect m7G modifications

  • RNA immunoprecipitation to identify methylated targets (e.g., Atf5 mRNA)

  • METTL1 interaction studies (co-immunoprecipitation, proximity labeling)

Key findings and considerations:

• TMEM11 deletion enhances cardiomyocyte proliferation and improves cardiac function after injury

• TMEM11 interacts with METTL1 and enhances m7G methylation of Atf5 mRNA

• The TMEM11-METTL1-ATF5-INCA1 axis forms a regulatory pathway for cardiomyocyte proliferation

• Expression pattern analysis shows TMEM11 increases during postnatal development, correlating with loss of regenerative capacity

How can researchers effectively analyze the assembly and dynamics of TMEM11-containing protein complexes?

TMEM11 forms multiple protein complexes of different molecular weights. Analyzing these complexes requires specialized techniques:

Methodological approaches:

• 2D Blue Native PAGE (BN-PAGE):

  • First dimension: Separation of intact protein complexes by molecular weight

  • Second dimension: SDS-PAGE for subunit analysis

  • Findings: TMEM11 assembles into complexes ranging from ~100 kDa to >1 MDa, with distinct enrichment at ~500 kDa (similar size to BNIP3/BNIP3L complexes)

• Complex stability analysis:

  • Differential detergent extraction to assess complex strength

  • Crosslinking prior to extraction for transient interactions

  • Salt extraction to distinguish integral membrane vs. peripheral membrane proteins

Interaction mapping:

• Proximity-based approaches:

  • APEX2 labeling for spatial proteomics

  • BioID for identifying proteins within defined cellular compartments

• Direct interaction assessment:

  • Yeast two-hybrid for binary interactions (has confirmed direct TMEM11-BNIP3/BNIP3L binding)

  • In vitro binding assays with recombinant proteins

Technical considerations:

• The choice of detergent significantly impacts complex stability

• TMEM11 predominantly forms smaller complexes than the MICOS complex

• Tag size can influence complex migration (e.g., APEX2-GFP-TMEM11 shifts complex size compared to untagged protein)

What approaches can be used to investigate the spatiotemporal regulation of mitophagy by TMEM11?

TMEM11 appears to regulate the spatial distribution of mitophagy sites rather than affecting the mitophagy process itself.

Advanced imaging techniques:

• Super-resolution microscopy:

  • SoRa confocal microscopy has been used to visualize TMEM11 relative to mitochondrial markers

  • STED or STORM microscopy for higher-resolution analysis of mitophagosome formation sites

• Live cell imaging:

  • Time-lapse fluorescence microscopy to track mitophagosome formation dynamics

  • Photoactivatable or photoconvertible fluorescent tags to track protein movement

Quantitative analysis frameworks:

• Mitophagosome quantification:

  • Number of BNIP3/BNIP3L-enriched structures per cell

  • Distribution patterns (clustered vs. dispersed)

  • Network connectivity (network-attached vs. isolated structures)

• Co-localization analysis:

  • Triple co-localization of TMEM11, BNIP3/BNIP3L, and LC3 at mitophagy sites

  • Key finding: TMEM11 co-enriches with BNIP3 at 70.9% and with BNIP3L at 65.4% of focal structures

Important research insights:

• TMEM11 depletion drastically increases the number of BNIP3-enriched mitophagy sites without altering their distribution pattern or mitochondrial network connectivity

• During CoCl₂-induced mitophagy, approximately 75% of mitophagosomes remain connected to the mitochondrial network regardless of TMEM11 presence

• TMEM11 does not affect incorporation of inner membrane proteins (e.g., MIC60) into mitophagosomes

What are the key considerations for using recombinant TMEM11 protein in experimental applications?

Recombinant TMEM11 protein is available for research applications, but several technical considerations should be addressed:

Production and purification:

• Expression systems: E. coli has been successfully used for producing recombinant TMEM11

• Purification tags: His6-ABP tag is commonly used for TMEM11

• Buffer conditions: PBS with 1M Urea, pH 7.4 has been reported for storage

Quality control parameters:

• Purity assessment: >80% by SDS-PAGE and Coomassie blue staining is typical

• Concentration determination: Expected concentration >0.5 mg/ml

• Functional validation: Antibody competition assays are a primary application

Experimental applications:

• Antibody validation/specificity confirmation

• Protein-protein interaction studies (requires proper refolding)

• Structure-function analysis of domains

Limitations and considerations:

• Recombinant proteins may lack post-translational modifications present in native proteins

• Membrane proteins are particularly challenging to express and fold correctly

• Hydrophobic transmembrane domains may require detergent solubilization for proper folding

What methodological approaches are recommended for studying TMEM11 in macrophage functionality?

Recent research has investigated TMEM11's role in macrophage function, particularly in immune responses:

Experimental models:

• Primary cell isolation:

  • Bone marrow-derived macrophages (BMDMs) from TMEM11 knockout mice

  • Protocols for proper differentiation and polarization

• Functional assays:

  • Macrophage polarization: M1 vs. M2 marker analysis (not affected by TMEM11 loss)

  • NLRP3 inflammasome activation: Reduced in TMEM11-deficient macrophages upon viral infection

  • Cytokine production: IL-1β and IL-18 (reduced in knockout)

  • Pyroptosis assessment: Reduced in TMEM11-deficient cells

Technical considerations:

• Mitochondrial morphology assessment is critical as a phenotypic readout

• Expression analysis of mitochondrial architecture proteins (MIC60, MIC10) and ETC complex proteins

• Viral infection models: VSV and IAV have been used to evaluate immune responses

Key findings:

• Unlike in Drosophila, TMEM11 loss in mouse macrophages does not affect expression of key mitochondrial architecture proteins

• TMEM11 is required for optimal NLRP3 inflammasome activation specifically during RNA virus infection

• Macrophage polarization is unaffected by TMEM11 loss, suggesting respiratory capabilities may be preserved despite altered mitochondrial morphology

How can researchers investigate the evolutionary conservation and divergence of TMEM11 function across species?

TMEM11 function shows both conservation and divergence across species, making comparative studies valuable:

Cross-species experimental approaches:

• Genetic complementation:

  • Expression of human TMEM11 in Drosophila PMI mutants to test functional rescue

  • Expression of Drosophila PMI in human TMEM11-depleted cells

• Sequence-structure-function analysis:

  • Alignment of TMEM11 orthologs across species (human, mouse, bovine, Drosophila)

  • Domain-focused mutational studies to identify critical functional regions

  • Structural modeling and prediction

Comparative phenotypic analysis:

• Drosophila PMI studies:

  • Enlarged mitochondria with abnormal cristae

  • Metabolic defects (reduced ATP production, increased ROS)

  • Shortened lifespan

• Mammalian TMEM11 studies:

  • Similar mitochondrial morphology defects

  • Cell type-specific functional consequences

  • Different impacts on mitochondrial protein expression compared to Drosophila

Methodological considerations:

• Use of standardized assays across species to enable direct comparisons

• Attention to species-specific differences in mitochondrial biology

• Comprehensive phenotypic analysis beyond just morphological assessment

This comparative approach can help identify core conserved functions versus species-specific adaptations of TMEM11.