Recombinant Mouse Myotubularin-related protein 10 (Mtmr10)

What is Myotubularin-related Protein 10 and which protein family does it belong to?

Myotubularin-related Protein 10 (Mtmr10) belongs to the myotubularin family, a large group of inositol polyphosphate 3-phosphatases. The myotubularin family consists of 16 different proteins, of which 9 members possess catalytic activity for dephosphorylating phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] at the D-3 position. The remaining 7 members, including Mtmr10, are catalytically inactive due to the absence of the conserved cysteine residue in the CX5R motif required for enzymatic activity . Mtmr10 is present in various tissues and is particularly enriched in brain tissue .

How does Mtmr10 expression vary across different tissue types?

Mtmr10 demonstrates variable expression across different tissues, with particularly high enrichment in brain tissue according to the Human Protein Atlas database . This tissue-specific expression pattern suggests that Mtmr10 may play specialized roles in neuronal function and maintenance. The differential expression of myotubularin family proteins across tissues is a common characteristic that contributes to their unique functions, despite sharing similar substrate preferences .

What are the structural characteristics of recombinant Mtmr10 protein?

Recombinant Mtmr10, like other myotubularin family proteins, contains specific structural domains that determine its function and interactions. While inactive myotubularins lack catalytic activity, they maintain important structural features that enable protein-protein interactions, particularly with catalytically active myotubularins. These structural characteristics include coiled-coil domains that facilitate dimerization with other myotubularin proteins . For recombinant protein preparation, these structural considerations are important for maintaining proper folding and functional interactions when expressed in heterologous systems.

How do inactive myotubularins like Mtmr10 regulate the function of active myotubularin family members?

While Mtmr10 is catalytically inactive, it likely plays a regulatory role through interactions with active myotubularin family members. This regulatory mechanism is observed with other inactive/active myotubularin pairs in the family. For example, MTMR9 (inactive) forms complexes with active members like MTMR6 and MTMR8, significantly altering their enzymatic activity and substrate specificity. The MTMR6/R9 complex shows a 30-fold increase in activity toward PtdIns(3,5)P2 but only a 2-fold increase toward PtdIns(3)P. Conversely, the MTMR8/R9 complex demonstrates a 4-fold increase in activity toward PtdIns(3)P but only a 1.4-fold increase toward PtdIns(3,5)P2 . By analogy, Mtmr10 may form similar regulatory complexes with catalytically active family members, modulating their activity, substrate preference, subcellular localization, and stability.

What neuronal functions does Mtmr10 regulate and through which molecular mechanisms?

Studies in C. elegans have shown that MTM-10 protects dendrites from degeneration caused by oxidative stress or Pseudomonas aeruginosa infection. MTM-10 is expressed in the AWC neurons of C. elegans and functions in a cell-autonomous manner to protect against pathogen-induced dendrite degeneration . The molecular mechanisms likely involve regulation of phosphoinositide signaling pathways, which are crucial for membrane trafficking, cytoskeletal organization, and cell survival. Since phosphoinositides serve as critical regulators of endocytic membrane trafficking , Mtmr10 may influence neuronal health by regulating these pathways through interactions with catalytically active myotubularins.

How does Mtmr10 contribute to disease pathogenesis when mutated or dysregulated?

While specific diseases directly linked to Mtmr10 mutations are not detailed in the provided search results, the myotubularin family is associated with various diseases. Mutations in both active and inactive myotubularins are linked to conditions such as myotubular myopathy and Charcot-Marie-Tooth disease . Given Mtmr10's enrichment in neural tissues and its protective role against dendrite degeneration observed in model organisms , its dysfunction could potentially contribute to neurodegenerative processes. The disease relevance of inactive myotubularins is highlighted by the fact that they can significantly alter the function of active partners, suggesting that Mtmr10 mutations could disrupt critical protein-protein interactions necessary for normal cellular function.

What are the critical factors to consider when designing experiments to study Mtmr10 function in neuronal systems?

When designing experiments to investigate Mtmr10 function in neuronal systems, researchers should consider:

• Expression system selection: Given that Mtmr10 is highly enriched in brain tissue , selection of appropriate neuronal cell lines or primary neuronal cultures is essential.

• Partner protein identification: Since inactive myotubularins function through interactions with active family members , co-immunoprecipitation experiments should be conducted to identify Mtmr10's binding partners.

• Stress conditions: As Mtmr10 appears to protect against oxidative stress and pathogen-induced damage , experiments should include appropriate stress conditions to evaluate its protective function.

• Temporal considerations: The dynamic nature of transcriptomes requires linking analysis to the specific biological state of samples under investigation .

• Statistical power: Adequate sampling of biological variation is essential, with power calculations incorporated into experimental design when possible .

What are the recommended protocols for expression and purification of recombinant mouse Mtmr10?

For optimal expression and purification of recombinant mouse Mtmr10, consider the following protocol guidelines:

• Expression system: Mammalian expression systems (such as HEK293 or CHO cells) are recommended for proper folding and potential post-translational modifications.

• Purification approach: Use of affinity tags (His or FLAG) followed by size exclusion chromatography to ensure purity and proper oligomeric state.

• Buffer conditions: Based on approaches used for other myotubularin family proteins, phosphate buffers with reducing agents are typically employed to maintain protein stability.

• Storage considerations: Similar to other recombinant proteins, lyophilization from a 0.2 μm filtered solution with a carrier protein like BSA can enhance stability and shelf-life .

• Quality control: Verification of protein identity and purity through mass spectrometry and SDS-PAGE analysis, with functional verification through binding assays with known interaction partners.

How should researchers design experiments to investigate Mtmr10 interactions with active myotubularin family members?

To effectively study Mtmr10 interactions with active myotubularin family members, researchers should:

• Co-immunoprecipitation studies: Following the approach used to demonstrate MTMR8/R9 and MTMR6/R9 interactions , use epitope-tagged proteins (HA-tagged active myotubularins and FLAG-tagged Mtmr10) for co-immunoprecipitation experiments.

• Protein stability assessments: Determine if the formation of complexes stabilizes the proteins by analyzing protein levels in cycloheximide-treated cells with and without co-expression of potential binding partners .

• Subcellular localization studies: Use fluorescently tagged proteins to determine if complex formation alters the subcellular localization of either protein partner.

• Enzymatic activity assays: For active partners, measure phosphatase activity toward different substrates (PtdIns(3)P and PtdIns(3,5)P2) in the presence and absence of Mtmr10 .

What statistical approaches are most appropriate for analyzing Mtmr10 expression data across different experimental conditions?

For analyzing Mtmr10 expression data, researchers should consider:

How can researchers effectively analyze the impact of Mtmr10 on phosphoinositide metabolism?

To analyze Mtmr10's impact on phosphoinositide metabolism, researchers should:

• Quantitative lipid analysis: Use methods like thin-layer chromatography or mass spectrometry to quantify cellular levels of phosphoinositides, particularly PtdIns(3)P and PtdIns(3,5)P2, in the presence or absence of Mtmr10.

• Visualization techniques: Employ phosphoinositide-specific antibodies for immunofluorescence to visualize changes in cellular distribution, counting spots larger than 1 nm as one PI(3)P molecule .

• Functional readouts: Assess downstream cellular processes regulated by phosphoinositides, such as autophagy (which can be inhibited by the MTMR8/R9 complex) or apoptosis (inhibited by the MTMR6/R9 complex) .

• Data normalization: Consider transfection efficiency (aim for >95%) when comparing phosphoinositide levels, and collect data from multiple cells (e.g., 50 cells per coverslip, three coverslips per condition) .

What cellular processes are regulated by Mtmr10 and how can these be experimentally measured?

Based on studies of related myotubularin family proteins, Mtmr10 may regulate several cellular processes:

• Neuronal protection: Given its protective role against dendrite degeneration , researchers can measure:

  • Dendrite morphology using fluorescent markers

  • Neuronal survival under stress conditions

  • Resistance to pathogen-induced damage

• Membrane trafficking: Through interaction with active myotubularins that regulate phosphoinositides critical for endocytic membrane trafficking , assess:

  • Endosome morphology and dynamics

  • Receptor internalization and recycling

  • Vesicular trafficking pathways

• Cell survival pathways: Similar to how the MTMR6/R9 complex inhibits apoptosis and the MTMR8/R9 complex inhibits autophagy , evaluate:

  • Apoptosis markers (caspase activation, PARP cleavage)

  • Autophagy markers (LC3 conversion, p62 levels)

  • Cell viability under various stress conditions

How can knockout or knockdown models be used to evaluate Mtmr10 function in vivo?

To evaluate Mtmr10 function in vivo using knockout or knockdown approaches:

• Generation of models: Create conditional knockout mice using Cre-lox system (particularly useful for tissue-specific deletion) or use CRISPR-Cas9 for targeted gene editing.

• Phenotypic analysis: Assess:

  • Neurological function (given Mtmr10's enrichment in brain tissue )

  • Response to oxidative stress or pathogen challenges

  • Developmental milestones, particularly in neural tissues

• Complementation studies: Perform rescue experiments with wild-type Mtmr10 or mutant variants to confirm specificity of observed phenotypes.

• Partner protein analysis: Examine how Mtmr10 deletion affects the expression, stability, and function of its interaction partners, similar to studies on MTMR9 knockout mice .

• Compensatory mechanisms: Investigate potential upregulation of other myotubularin family members that might compensate for Mtmr10 loss.

What are the potential therapeutic implications of targeting Mtmr10 interactions in neurological disorders?

Given Mtmr10's role in protecting against neuronal degeneration , targeting its interactions could have therapeutic potential:

• Neuroprotective approaches: Enhancing Mtmr10's protective function could potentially mitigate neurodegeneration induced by oxidative stress or pathogen exposure.

• Protein-protein interaction modulators: Developing small molecules that enhance favorable interactions between Mtmr10 and its active partners might boost protective signaling pathways.

• Phosphoinositide pathway targeting: Since myotubularin family proteins regulate phosphoinositide metabolism , which affects numerous cellular processes, modulation of these pathways could have broad therapeutic applications.

• Disease-specific applications: While no specific diseases are directly linked to Mtmr10 mutations in the provided search results, its enrichment in brain tissue suggests potential relevance to neurological disorders.

How does the function of Mtmr10 compare across different model organisms and what are the implications for translational research?

Comparing Mtmr10 function across model organisms provides insights for translational research:

• C. elegans findings: Studies in C. elegans show MTM-10 protects against dendrite degeneration induced by oxidative stress or pathogen infection, functioning cell-autonomously in AWC neurons .

• Mammalian systems: While specific mouse Mtmr10 functions aren't detailed in the search results, the high conservation of myotubularin family proteins suggests similar protective roles may exist in mammals.

• Evolutionary conservation: The myotubularin family is well-conserved across species, suggesting fundamental roles in cellular physiology that may translate across models to humans.

• Translational considerations: When moving from simple models to complex mammalian systems, researchers should account for:

  • Tissue-specific expression patterns

  • More complex interaction networks

  • Potential redundancy among family members

  • Species-specific differences in phosphoinositide metabolism