Recombinant Rat Transmembrane protein 11, mitochondrial (Tmem11)

What expression systems are commonly used for recombinant rat TMEM11 production?

Based on commercial research products, recombinant rat TMEM11 is successfully expressed in both prokaryotic and eukaryotic systems:

• E. coli expression system - Used for producing full-length rat TMEM11 (1-190aa) with His-tag modifications

• HEK293 cells - Employed for producing recombinant rat TMEM11 that maintains proper protein folding and post-translational modifications

The choice between these systems depends on research requirements, with E. coli providing higher yields while HEK293 cells offer better functional properties.

How does TMEM11 regulate mitochondrial morphology?

TMEM11 is a critical regulator of mitochondrial network architecture. Depletion of TMEM11 through siRNA or CRISPRi techniques leads to dramatic alterations in mitochondrial morphology:

• In control cells with normal TMEM11 expression, approximately 90% display tubular mitochondria filling the cytoplasm

• Upon TMEM11 knockdown, up to 45% of cells exhibit spherical and enlarged mitochondria, described as a "balloon phenotype"

• TMEM11-depleted cells consistently show more than half of their mitochondria becoming enlarged and/or bulbous compared to the narrow tubular mitochondria observed in control cells

These morphological changes are distinct from those seen with depletion of other mitochondrial dynamics proteins like DRP1 (causing elongated tubules with some spherical entities) or OPA1 (causing small, numerous spherical mitochondria) . This suggests TMEM11 has a unique role in maintaining proper mitochondrial architecture.

What molecular mechanisms underlie TMEM11's role in cardiomyocyte proliferation?

TMEM11 regulates cardiomyocyte proliferation through a complex molecular pathway:

• TMEM11 directly interacts with METTL1 (methyltransferase-like protein 1)

• This interaction enhances m7G methylation of Atf5 mRNA, thereby increasing ATF5 protein expression

• Elevated ATF5 promotes the transcription of Inca1, an inhibitor of cyclin-dependent kinase interacting with cyclin A1

• INCA1 suppresses cardiomyocyte proliferation through cell cycle regulation

Experimental evidence shows that TMEM11 deletion enhances cardiomyocyte proliferation and improves heart function after myocardial injury. Conversely, TMEM11 overexpression inhibits neonatal cardiomyocyte proliferation and regeneration in mouse hearts . This TMEM11-METTL1-ATF5-INCA1 axis represents a potential therapeutic target for promoting cardiac repair and regeneration.

How does TMEM11 function in mitophagy regulation?

TMEM11 acts as a negative regulator of receptor-mediated mitophagy:

• It localizes to the outer mitochondrial membrane where it directly interacts with the mitophagy receptors BNIP3 and BNIP3L (NIX)

• TMEM11 stably forms a complex with BNIP3/BNIP3L that inhibits their mitophagy-promoting function

• Depletion of TMEM11 enhances both basal mitophagy levels and hypoxia-induced mitophagy

• This effect is specific to mitophagy, as basal macroautophagy (non-selective autophagy) remains unaltered by TMEM11 depletion

Under hypoxic conditions or with hypoxia mimetics like CoCl2, TMEM11 depletion further enhances BNIP3-dependent mitophagy, suggesting that metabolic state changes critically impact the TMEM11-NIX engagement dynamics . This positions TMEM11 as a key player in the fine-tuning of mitochondrial quality control.

What techniques are recommended for studying TMEM11's interaction with the MICOS complex?

To effectively study TMEM11's relationship with the Mitochondrial Contact Site and Cristae Organizing System (MICOS), researchers should consider these methodological approaches:

• Proteomic analysis - To identify protein-protein interactions within the MICOS/MIB complex

• Co-immunoprecipitation - To confirm direct interactions between TMEM11 and MICOS components

• CRISPRi - For specific gene depletion to study functional relationships

• Blue native PAGE - To analyze the assembly of protein complexes

• Super-resolution microscopy - To visualize TMEM11 localization relative to MICOS components

What experimental approaches can assess TMEM11's role in cardiac regeneration?

Investigating TMEM11's role in cardiac regeneration requires a multi-faceted approach:

• Genetic manipulation models:

  • TMEM11 knockout mice to assess enhanced regenerative capacity

  • TMEM11 overexpression models to confirm inhibitory effects

  • Targeted manipulation of the TMEM11-METTL1-ATF5-INCA1 pathway

• Myocardial injury models:

  • Myocardial infarction (MI) induction followed by assessment of cardiac function

  • Evaluation of heart function after injury using echocardiography

  • Histological analysis of cardiac tissue repair

• Cell proliferation assays:

  • Measurement of cardiomyocyte proliferation markers

  • Cell cycle analysis in isolated cardiomyocytes

  • EdU incorporation assays to quantify DNA synthesis

These approaches provide comprehensive evaluation of how TMEM11 impacts cardiac repair processes and offer insights into potential therapeutic interventions targeting the TMEM11 pathway for heart failure treatment.

What are the recommended storage and handling conditions for recombinant rat TMEM11?

For optimal stability and functionality of recombinant rat TMEM11:

• TMEM11 lyophilized powder:

  • Store at -20°C/-80°C upon receipt

  • Aliquot after reconstitution to avoid repeated freeze-thaw cycles

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

• TMEM11 pre-coupled magnetic beads:

  • Store at 2-8°C

  • Do not freeze-thaw the beads

  • Stable for at least 6 months under proper storage conditions

  • Maintained in PBS buffer at 10mg beads/mL

Repeated freezing and thawing is not recommended for either format. For working aliquots of protein preparations, storage at 4°C for up to one week is acceptable .

What techniques can distinguish between TMEM11's inner and outer mitochondrial membrane functions?

Given the emerging evidence of TMEM11's dual localization, these techniques can help differentiate its membrane-specific functions:

• Submitochondrial fractionation with protease protection assays:

  • Separate inner and outer mitochondrial membranes

  • Use protease sensitivity as a marker for membrane localization

  • Compare protein degradation patterns with known IMM and OMM markers

• Domain-specific antibodies or tagged constructs:

  • Generate tools targeting specific regions of TMEM11

  • Track different portions of the protein in distinct mitochondrial compartments

• Site-directed mutagenesis:

  • Create TMEM11 variants with altered membrane targeting sequences

  • Assess localization and function of these mutants

• Functional assays with membrane-specific inhibitors:

  • Use compounds that selectively permeabilize specific mitochondrial membranes

  • Measure TMEM11-dependent functions under these conditions

These approaches help resolve the apparent contradiction between earlier studies localizing TMEM11 to the IMM and recent findings of its OMM localization and function .

What considerations are important when using TMEM11 pre-coupled magnetic beads for protein interaction studies?

When utilizing TMEM11 pre-coupled magnetic beads for investigating protein interactions:

• Bead properties:

  • Particle size: ~2 μm

  • Surface: Hydrophilic

  • Capacity: > 200 pmol rabbit IgG/mg beads

• Experimental considerations:

  • Optimize buffer conditions to maintain protein-protein interactions

  • Include appropriate controls (non-specific protein-coupled beads)

  • Consider pre-clearing samples to reduce non-specific binding

  • Use gentle washing techniques to preserve weak interactions

• Applications:

  • Immunoprecipitation/Co-precipitation

  • Protein/antibody separation and purification

  • Cell sorting

  • High-throughput operations with automation equipment

This pre-coupled format allows for convenient and fast capture of target molecules with high specificity and efficient magnetic separation for studying TMEM11's diverse interaction partners.

How does the functional relationship between TMEM11 and BNIP3/BNIP3L change under metabolic stress?

The TMEM11-BNIP3/BNIP3L interaction is highly sensitive to metabolic conditions:

• Under basal conditions:

  • TMEM11 maintains a steady inhibitory effect on BNIP3/BNIP3L-mediated mitophagy

  • This ensures appropriate levels of mitochondrial turnover for normal cellular function

• Under hypoxic conditions:

  • TMEM11 depletion enhances BNIP3-dependent mitophagy more dramatically than under basal conditions

  • Hypoxia mimetics like CoCl₂ amplify this effect, suggesting oxygen-sensing mechanisms are involved

• Metabolic stress conditions:

  • May alter the temporal dynamics of TMEM11-NIX engagement

  • Could involve post-translational modifications of either protein

  • Potentially engage additional regulatory proteins that modulate the interaction

Future research should explore whether TMEM11 functions as a metabolic sensor, either through its association with MICOS/MIB within mitochondria or via its interaction with NIX at the peri-mitochondrial environment. Understanding this relationship would provide crucial insights into how cells adapt mitochondrial quality control to changing metabolic demands.

What is the potential of targeting the TMEM11-METTL1-ATF5-INCA1 axis for cardiac regeneration therapies?

The TMEM11-regulated pathway presents a promising therapeutic target for cardiac regeneration:

• Therapeutic potential:

  • TMEM11 inhibition could enhance endogenous cardiomyocyte proliferation

  • This approach might promote cardiac repair after myocardial injury

  • Targeting this pathway could overcome the limited regenerative capacity of the adult heart

• Intervention strategies:

  • Small molecule inhibitors of TMEM11-METTL1 interaction

  • RNA-based therapies to modulate TMEM11 expression

  • Gene therapy approaches to temporarily suppress TMEM11 function in damaged cardiac tissue

• Translational considerations:

  • Temporal control of intervention to promote repair without disrupting normal cardiac function

  • Tissue-specific targeting to avoid systemic effects on mitochondrial dynamics

  • Balance between enhanced proliferation and maintaining cardiomyocyte maturity and function

Research suggests that transient modulation of this pathway after cardiac injury could provide a novel therapeutic strategy that harnesses endogenous regenerative mechanisms rather than relying on cell transplantation approaches .

How might TMEM11's dual roles in mitochondrial morphology and mitophagy be integrated into a unified model?

A comprehensive model of TMEM11 function must account for its effects on both mitochondrial morphology and mitophagy:

• Structural role:

  • TMEM11 at the IMM contributes to cristae organization and mitochondrial network architecture

  • Depletion leads to distinctive "balloon" morphology of mitochondria

• Regulatory role:

  • TMEM11 at the OMM interacts with BNIP3/BNIP3L to inhibit mitophagy

  • Functions as a quality control checkpoint for mitochondrial turnover

• Integrated model:

  • TMEM11 may serve as a bridge between mitochondrial structure and turnover decisions

  • Structural changes in mitochondria could influence TMEM11's distribution between IMM and OMM

  • This redistribution could modulate its interaction with mitophagy receptors

  • The process potentially creates a feedback loop where mitochondrial morphology changes trigger appropriate mitophagy responses

Understanding how these dual functions are coordinated may reveal fundamental principles about mitochondrial quality control mechanisms and provide insights into disorders characterized by mitochondrial dysfunction.