Recombinant Mouse Transmembrane protein 170A (Tmem170a)

What is the basic structure of TMEM170A?

TMEM170A is a small transmembrane protein (approximately 15.25 kDa in humans) containing three transmembrane domains. According to TMPRED software predictions, the N-terminus is likely luminal while the C-terminus is cytoplasmic . Experimental validation using TMEM170A-GFP fusion proteins confirmed this topology, as the GFP tag at the C-terminus was accessible to anti-GFP antibodies in digitonin-permeabilized cells (which permeabilizes only the plasma membrane), while luminal ER proteins required additional Triton X-100 permeabilization to be detected .

Where is TMEM170A localized in mammalian cells?

TMEM170A primarily localizes to the endoplasmic reticulum (ER) and nuclear envelope membranes. This has been validated through multiple tagged constructs including TMEM170A-GFP, FLAG-TMEM170A, and myc-TMEM170A, all of which demonstrate consistent localization patterns to both peripheral ER and nuclear envelope membranes . Co-localization studies with established ER markers such as calnexin and RTN4 further confirm this distribution pattern .

How conserved is TMEM170A across species?

TMEM170A is highly conserved across major eukaryotic phyla, suggesting an evolutionarily important function . While specific conservation data for the mouse version is not explicitly detailed in the available sources, the human TMEM170A (UniProtKB/Swiss-Prot accession Q8WVE7) has been well-characterized, and conservation analysis indicates significant homology across species boundaries .

What approaches can be used to study TMEM170A expression and localization?

Several complementary approaches have proven effective for studying TMEM170A:

• Fluorescent protein fusion constructs: Creating TMEM170A fusion constructs with GFP, FLAG, or myc tags enables visualization via fluorescence microscopy. For optimal results, both transient transfection and stable cell line generation have been successfully employed .

• Immunofluorescence microscopy: Using antibodies against TMEM170A or epitope tags in fixed cells. This approach allows co-localization studies with other cellular markers .

• Immunoblot analysis: For quantification of protein expression levels and validation of knockdown efficiency .

• Electron microscopy: For high-resolution analysis of subcellular localization and effects on membrane structures .

What are effective methods to manipulate TMEM170A expression levels?

Researchers have successfully employed multiple approaches to alter TMEM170A expression:

• RNA interference: siRNA-mediated silencing has been effectively used to downregulate TMEM170A. For optimal knockdown, transfection with TMEM170A-specific siRNAs at appropriate concentrations (typically in the nanomolar range) for 48-72 hours has yielded greater than 80% reduction in protein levels .

• Overexpression systems: Transient transfection with plasmids encoding TMEM170A (with or without tags) under strong promoters has been used to achieve overexpression. Both transient transfection and stable cell line generation approaches are viable depending on experimental needs .

• Stable cell lines: Generation of cell lines stably expressing TMEM170A fusion proteins provides consistent expression levels for long-term studies .

How can interactions between TMEM170A and other proteins be studied?

Several techniques have been employed to investigate TMEM170A's interactions:

• Co-immunoprecipitation: For identifying physical interactions with other proteins, such as the documented interaction with RTN4 .

• Immunofluorescence co-localization: To assess spatial proximity with other proteins in fixed cells .

• Proximity ligation assays: While not explicitly mentioned in the provided sources, this technique would be appropriate for detecting protein-protein interactions in situ.

• Co-expression studies: Examining phenotypic outcomes when TMEM170A is co-expressed with interacting partners has revealed functional relationships, as demonstrated with RTN4 .

How does TMEM170A affect ER structure and organization?

TMEM170A plays a critical role in determining ER morphology, particularly in regulating the balance between tubular ER and ER sheets:

• Downregulation effects: Silencing TMEM170A induces the formation of tubular ER, often confined to restricted areas asymmetrically distributed around the nucleus. This effect was confirmed by both immunofluorescence microscopy and high-resolution electron microscopy .

• Overexpression effects: Conversely, overexpression of TMEM170A promotes the formation and expansion of ER sheets. Ultrastructural analysis revealed extensive, well-organized ER sheet stacks decorated with membrane-bound ribosomes in cells overexpressing TMEM170A .

• ER sheet marker effects: TMEM170A silencing reduces the levels of CLIMP-63, an ER-sheet-specific marker protein, to approximately 28% of control levels (p=6.89×10⁻⁵), further supporting its role in ER sheet formation .

What is the relationship between TMEM170A and reticulon proteins in ER morphogenesis?

TMEM170A and reticulon proteins (particularly RTN4) exhibit antagonistic functions in ER morphogenesis:

• Opposing actions: While reticulons promote ER tubule formation, TMEM170A promotes ER sheet formation .

• Physical interaction: TMEM170A has been shown to physically interact with RTN4, suggesting direct functional interplay between these proteins with opposing effects on ER morphology .

• Antagonistic relationship: When co-expressed, the phenotypic outcomes suggest that TMEM170A and RTN4 counteract each other's effects on ER morphology, nuclear envelope formation, and nuclear pore complex assembly .

What phenotypic changes are observed in the ER when TMEM170A expression is altered?

Detailed microscopic and ultrastructural analyses have revealed distinct phenotypes:

These observations confirm TMEM170A as an ER-sheet-promoting protein whose cellular concentration directly affects the balance between tubular ER and ER sheets .

How does TMEM170A affect nuclear envelope structure and nuclear morphology?

TMEM170A manipulation significantly impacts nuclear envelope architecture:

• Nuclear shape alterations: TMEM170A silencing induces nuclear envelope invaginations and evaginations, resulting in altered nuclear shape .

• Nuclear size effects: Downregulation of TMEM170A increases nuclear surface area to approximately 141% of control cells (641.82±28.2 μm² in controls vs. 906.58±36.52 μm² in silenced cells, p=0.00019) and nuclear volume to approximately 137% of control cells (867.91±16.51 μm³ in controls vs. 1192.4±28.39 μm³ in silenced cells, p=6.84×10⁻⁵) .

• Opposing effects with overexpression: Conversely, TMEM170A overexpression reduces nuclear surface area to approximately 84% of control cells (622.21±6.87 μm² in controls vs. 522.14±15.88 μm² in overexpressing cells) .

What is the effect of TMEM170A on nuclear pore complexes (NPCs)?

TMEM170A plays a crucial role in NPC formation and distribution:

• NPC density changes: TMEM170A silencing significantly decreases the density of nuclear pore complexes in the nuclear envelope, as visualized by both immunofluorescence and electron microscopy .

• Nucleoporin localization: Downregulation of TMEM170A affects the proper localization of nucleoporins, components of the NPC, suggesting a role in NPC assembly or maintenance .

• Mechanistic relationship: The exact mechanism by which TMEM170A influences NPC formation remains under investigation, but may relate to its effects on nuclear envelope structure and/or interactions with other nuclear envelope proteins .

How does TMEM170A affect inner nuclear membrane (INM) protein distribution?

TMEM170A plays a critical role in the proper localization of INM proteins:

• Reduced nuclear rim localization: TMEM170A silencing reduces the nuclear rim signal of INM proteins such as LAP2β and emerin .

• Mislocalization to ER: In TMEM170A-depleted cells, LBR (lamin B receptor) prominently mislocalizes from the INM to the ER in more than 80% of cells, and forms striking ER-associated aggregates in nearly 20% of cells .

• Co-aggregation with ER markers: These LBR-containing aggregates in the ER also contain the ER marker protein calnexin, confirming their ER localization .

What are the optimal conditions for producing recombinant mouse TMEM170A protein?

While the search results don't specifically address recombinant mouse TMEM170A production, we can extrapolate from standard approaches and related protein production methods:

• Expression systems: Based on protocols for similar transmembrane proteins, insect cell expression systems (such as Sf21 cells with baculovirus) might be appropriate for producing properly folded TMEM170A, as demonstrated for other multi-pass membrane proteins .

• Purification considerations: Being a transmembrane protein, detergent solubilization and specialized chromatography techniques would likely be required for purification. Carrier-free formulations may be prepared similarly to other recombinant membrane proteins by lyophilization from filtered PBS solutions .

• Stability and storage: Given the stability challenges with membrane proteins, addition of stabilizing agents may be necessary, though carrier-free versions without BSA would be valuable for applications where BSA might interfere with experiments .

What experimental approaches can distinguish between the direct and indirect effects of TMEM170A on cellular structures?

Several sophisticated approaches can help delineate direct versus indirect effects:

• Domain mapping and mutagenesis: Creating truncated versions or point mutations in TMEM170A can help identify which domains are essential for specific functions, distinguishing between different aspects of its activity .

• Rescue experiments: Silencing endogenous TMEM170A followed by expression of siRNA-resistant variants (wild-type or mutated) can help establish causality and domain-specific functions .

• Temporal analysis: Time-course experiments after induction or repression of TMEM170A expression can help establish the sequence of events and distinguish primary from secondary effects.

• Interactome analysis: Comprehensive identification of TMEM170A-interacting proteins through approaches like BioID or proximity labeling would help establish the molecular network through which TMEM170A exerts its functions .

How can researchers quantitatively assess TMEM170A's effects on ER and nuclear envelope morphology?

Quantitative approaches to assess morphological changes include:

• Morphometric analysis: Nuclear surface area and volume measurements using 3D microscopy techniques have already yielded quantitative data on nuclear changes (as detailed in section 4.1) .

• ER tubule/sheet ratio quantification: Quantitative electron microscopy approaches can measure the relative abundance of tubular versus sheet ER structures under different TMEM170A expression conditions .

• Marker protein quantification: Western blot quantification of sheet-specific marker proteins like CLIMP-63 (reduced to 28.47±2.25% in TMEM170A-silenced cells) provides molecular correlates of morphological changes .

• NPC density measurements: Quantitative immunofluorescence or electron microscopy can assess NPC density per unit area of nuclear envelope, providing a numerical measure of TMEM170A's effect on NPC formation .