Recombinant Danio rerio Transmembrane protein 242 (tmem242)

Where is tmem242 localized in zebrafish cells?

Studies using subcellular fractionation and immunofluorescence microscopy have demonstrated that tmem242 is exclusively localized to the mitochondria, specifically as an integral component of the inner mitochondrial membrane (IMM) . The protein's topology shows that both N- and C-terminal regions face the mitochondrial matrix. This localization is consistent with its functional role in mitochondrial processes, particularly in the assembly of ATP synthase components .

How is the expression of tmem242 regulated during zebrafish development?

While detailed developmental expression patterns aren't fully characterized in the provided search results, RNAseq analysis has identified tmem242 as one of 89 transmembrane protein genes expressed in zebrafish thrombocytes . This suggests developmental regulation related to blood cell differentiation. Knockdown experiments in both adult zebrafish and 3-day post-fertilization larvae demonstrate that the gene is functionally expressed at multiple developmental stages, allowing for analysis of its role throughout development .

What is the primary function of tmem242 in zebrafish?

The primary function of tmem242 in zebrafish appears to be modulating the assembly of ATP synthase in the mitochondrial inner membrane . It serves as an assembly factor for the c8-ring, which provides the membrane sector of the enzyme's rotor. This role is critical for proper ATP synthase function and consequently affects cellular energy production. Additionally, tmem242 influences reactive oxygen species (ROS) levels, functioning as a ROS modulator, with its absence leading to elevated ROS production . Through these mechanisms, tmem242 indirectly affects coagulation processes and hemostasis.

How does tmem242 knockdown affect hemostasis in zebrafish?

Knockdown of tmem242 in zebrafish results in significantly increased bleeding as demonstrated by gill bleeding assays . This hemostatic defect is primarily attributed to impaired coagulation rather than defects in thrombocyte production or aggregation. Specifically:

• Thrombocyte counts remain unchanged in tmem242 knockdown zebrafish

• Whole blood aggregation responses to ADP and collagen are unaffected

• Both kinetic partial thromboplastin time (kPTT) and kinetic prothrombin time (kPT) assays show delayed fibrin formation

• Laser-induced venous thrombosis experiments reveal prolonged time-to-occlusion (TTO)

• Microthrombi formation is observed in larvae after tmem242 knockdown

These findings suggest a complex role for tmem242 in regulating coagulation processes, potentially through a mechanism similar to disseminated intravascular coagulation (DIC) .

What is the relationship between tmem242 and the ATP synthase complex?

Tmem242 is intimately involved in the assembly of the ATP synthase complex, particularly the c8-ring component of the rotor . Research has shown that:

• TMEM242 helps assemble the rotor ring of ATP synthase

• Deletion of TMEM242 affects but does not completely eliminate ATP synthase assembly

• TMEM242 interacts with subunit c of ATP synthase

• TMEM242 works in coordination with another transmembrane protein, TMEM70, in the assembly process

• Deletion of both TMEM70 and TMEM242 together completely prevents assembly of ATP synthase

Additionally, tmem242 influences the incorporation of subunits ATP6, ATP8, j, and k into the enzyme complex, a function not shared by TMEM70 . This ATP synthase assembly role directly connects to cellular energy production and ROS generation.

What are the most effective methods for knockdown of tmem242 in zebrafish?

Based on published research, the most effective approach for tmem242 knockdown in zebrafish involves antisense oligonucleotide (ASO) technology using the "piggyback" method . The protocol includes:

• Design of gene-specific ASOs that target tmem242 mRNA

• Hybridization of these ASOs with control vivo-morpholino (cVMO)

• Intravenous injection of the hybrid molecules into adult zebrafish

• Verification of knockdown efficiency by qRT-PCR (>95% knockdown has been achieved)

For larval studies, the same technology can be applied with adjustment of doses for the developmental stage. This method has proven effective for studying both acute and extended effects of tmem242 deficiency in zebrafish .

How can researchers measure ROS levels following tmem242 manipulation?

To assess ROS production after tmem242 knockdown in zebrafish, researchers can use the following protocol :

• Treat zebrafish larvae with 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) dye

• Allow the dye to permeate tissues and react with ROS

• Visualize the resulting green fluorescence using fluorescence microscopy

• Quantify ROS signals by measuring green fluorescence intensity using image analysis software (e.g., ImageJ)

Control experiments should include treatment with known ROS inhibitors (e.g., N-acetylcysteine) to validate the specificity of the signal. This methodology has successfully demonstrated significant increases in ROS production in tmem242 knockdown zebrafish larvae compared to controls .

What coagulation assays are suitable for evaluating the effects of tmem242 knockdown?

To comprehensively assess coagulation status following tmem242 knockdown, a combination of in vitro and in vivo assays has proven effective :

In vitro assays:

• Kinetic Partial Thromboplastin Time (kPTT) – Measures the intrinsic coagulation pathway using plasma from control and knockdown zebrafish

• Kinetic Prothrombin Time (kPT) – Evaluates the extrinsic coagulation pathway

• Western blot analysis of coagulation factors (particularly F9a) in plasma samples

In vivo assays:

• Gill bleeding assay – Quantifies bleeding tendency in adult zebrafish

• Laser-induced venous thrombosis – Measures time-to-occlusion (TTO) in larvae

• O-Dianisidine staining – Detects microthrombi formation in larvae

Molecular assays:

• qRT-PCR analysis of coagulation factor gene expression (f5, f7, f8, f9a, f9b, f9l, f10, tf, and vwf)

This multi-modal approach provides comprehensive insights into the specific coagulation defects resulting from tmem242 deficiency .

How does tmem242 knockdown affect reactive oxygen species (ROS) production and what are the downstream consequences?

The knockdown of tmem242 leads to increased ROS production through the following mechanistic pathway :

• Tmem242 deficiency impairs proper assembly of ATP synthase

• Impaired ATP synthase function leads to electron leakage from the respiratory chain

• This leakage results in enhanced ROS generation

The downstream consequences of this elevated ROS include:

• Activation of oxidative stress response pathways

• Upregulation of sirt6 and nrf2 transcription factors

• Altered expression of multiple hepatocyte transcription factors

• Significantly increased expression of coagulation factor genes, especially f9a (>10-fold increase)

• Paradoxical bleeding phenotype despite elevated coagulation factors, resembling disseminated intravascular coagulation (DIC)

This ROS-mediated pathway has been experimentally validated by demonstrating that:

• Inhibition of ATP synthase with oligomycin similarly elevates ROS and f9a transcripts

• ROS inhibition reduces f9a expression, confirming ROS-mediated regulation

• Knockdown of sirt6 and nrf2 reduces f9a levels, placing them in the regulatory pathway

What transcription factors are modulated by tmem242 knockdown and how do they affect coagulation factor expression?

Tmem242 knockdown has profound effects on multiple hepatocyte transcription factors that regulate coagulation gene expression. The following changes have been observed :

Decreased mRNA levels:

• gata6, zeb1b, foxo1a, foxo3b, snail1, zbtb20, creg1, spen, slug, and shp

Increased mRNA levels:

• foxa2, foxa3, sirt6, nrf2, bach1b, smad7, and bcl6b

Unchanged mRNA levels:

• foxa1, gata4, prox1a, cebp, foxo1b, foxo3a, hnf1a, hnf4a, and creb1b

The increased sirt6 and nrf2 appear particularly important, as:

• Their expression correlates with elevated f9a levels

• Knockdown of sirt6 and nrf2 reduces f9a expression

• They form part of the regulatory pathway connecting ROS production to coagulation factor expression

This transcriptional network explains how tmem242 deficiency ultimately affects hemostasis through altered coagulation factor expression .

How does tmem242 interact with other proteins in the mitochondrial respiratory chain?

Tmem242 has several important protein interactions within the mitochondrial respiratory system :

• Interaction with ATP synthase components:

  • Direct interaction with subunit c of ATP synthase

  • Cooperation with TMEM70 in assembling the c8-ring of ATP synthase

  • Involvement in incorporating subunits ATP6, ATP8, j, and k into the enzyme complex

• Interaction with respiratory complexes:

  • TMEM242 deletion affects the assembly of respiratory enzyme complexes I, III, and IV

  • Interaction with the mitochondrial complex I assembly (MCIA) complex

  • Combined deletion of TMEM70 and TMEM242 enhances the impact on complex I

• Functional protein interactions:

  • RNF5 and GGT7 have been identified as interaction partners of TMEM242

These interactions position tmem242 as an important coordinator of mitochondrial respiratory chain assembly and function, explaining its broad impact on cellular energy production and ROS generation when depleted .

How can tmem242 knockdown be used as a model for studying disseminated intravascular coagulation (DIC)?

The tmem242 knockdown zebrafish presents a unique model for studying DIC-like conditions based on the following observed features :

• Paradoxical hemostatic profile:

  • Enhanced bleeding despite elevated coagulation factor levels

  • Presence of microthrombi in larvae

  • Delayed clot formation in coagulation assays

• Experimental advantages of the model:

  • Temporal control of knockdown using inducible systems

  • Ability to study both adult and larval stages

  • Optical transparency of larvae allowing real-time visualization of thrombus formation

  • Established methodologies for quantifying bleeding and coagulation

• Research applications:

  • Screening potential therapeutic interventions for DIC

  • Studying the progression from hypercoagulability to consumption coagulopathy

  • Investigating the role of ROS in triggering coagulation disorders

  • Examining the transcriptional regulation of coagulation factor genes under pathological conditions

When using this model, researchers should include appropriate controls, including partial knockdowns to simulate different severities of the condition, and comparative analyses with established DIC models .

What are the methodological considerations for working with recombinant Danio rerio tmem242 protein?

When working with recombinant tmem242 protein, researchers should consider the following technical aspects :

• Expression systems:

  • E. coli-based expression systems have been successfully used

  • The recombinant protein is typically produced with tags (His, FLAG, Strep II) to facilitate purification

• Protein characteristics:

  • Full-length protein (142 amino acids) or specific domains can be expressed

  • Proper folding is critical for functional studies, especially for transmembrane domains

• Storage and handling:

  • Store at -20°C/-80°C for extended storage

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be maintained at 4°C for up to one week

  • Typical storage buffer contains Tris/PBS-based buffer with 6-50% Trehalose or glycerol, pH 8.0

• Reconstitution:

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

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

• Application considerations:

  • For structural studies, attention to the transmembrane nature of the protein is essential

  • For functional studies, incorporation into liposomes or nanodiscs may be necessary

  • For interaction studies, detergent selection is critical to maintain native-like folding

How can researchers verify the specificity of phenotypes observed in tmem242 knockdown studies?

To ensure that observed phenotypes are specifically due to tmem242 deficiency rather than off-target effects, researchers should implement the following validation strategies :

• Knockdown verification:

  • Quantify tmem242 mRNA levels by qRT-PCR (>95% knockdown efficiency is optimal)

  • Confirm protein depletion by Western blot when antibodies are available

• Control experiments:

  • Use scrambled/mismatched ASOs as negative controls

  • Include rescue experiments by co-expressing knockdown-resistant tmem242 constructs

• Multiple knockdown techniques:

  • Compare phenotypes between morpholino knockdown and CRISPR/Cas9 genome editing

  • Use inducible knockdown systems to establish temporal relationships

• Dose-response relationships:

  • Perform partial knockdowns to establish correlation between tmem242 levels and phenotype severity

• Cross-species validation:

  • Compare phenotypes with TMEM242-deficient mammalian cell lines or mouse models

  • Perform complementation studies with human TMEM242 in zebrafish knockdowns

• Pathway validation:

  • Manipulate downstream targets (e.g., ROS levels, sirt6, nrf2) to determine if they recapitulate or rescue phenotypes

  • Use specific inhibitors (e.g., oligomycin for ATP synthase, N-acetylcysteine for ROS) to confirm the proposed mechanisms

What are the key experimental findings regarding tmem242 knockdown effects on coagulation factors?

The following table summarizes the effects of tmem242 knockdown on coagulation factor gene expression in zebrafish :

This pattern of increased coagulation factor expression (except f8) despite a bleeding phenotype supports the hypothesis of a DIC-like condition where coagulation factors are being consumed in microthrombi formation .

How does tmem242 knockdown affect ATP synthase and respiratory chain complexes?

The impact of tmem242 deficiency on mitochondrial complexes has been characterized with the following findings :

These findings position tmem242 as a critical factor in the coordinated assembly of multiple respiratory chain components, explaining how its deficiency impacts cellular energetics and ROS production .

What transcription factor changes mediate the effects of tmem242 knockdown?

The complex transcriptional response to tmem242 knockdown involves multiple hepatocyte transcription factors, as summarized below :

Experimental validation has confirmed the particular importance of sirt6 and nrf2 in this pathway, as their knockdown reduces f9a expression levels, establishing them as critical intermediates in the ROS-mediated regulation of coagulation factors following tmem242 depletion .