Recombinant Chicken Mitochondrial Rho GTPase 1 (RHOT1), partial

What is chicken RHOT1 and how is it structurally organized?

Chicken Mitochondrial Rho GTPase 1 (RHOT1), also known as Miro1, is a mitochondrial outer membrane protein that belongs to the Ras Homolog Gene Family. The protein contains characteristic domains similar to mammalian RHOT1, including two GTPase domains separated by two EF-hand calcium-binding motifs and a C-terminal transmembrane domain. RHOT1 is evolutionarily conserved across species, with the functional domains showing high sequence homology between chicken and mammals. The protein plays crucial roles in mitochondrial trafficking, dynamics, and quality control mechanisms. For recombinant expression, researchers typically work with partial constructs focusing on specific functional domains, such as the N-terminal GTPase domain or the calcium-binding EF-hand domains, as these are critical for the protein's regulatory functions .

How do I confirm the identity and activity of purified recombinant chicken RHOT1?

Verification of recombinant chicken RHOT1 identity requires a multi-step approach. Western blotting using anti-RHOT1 antibodies provides preliminary confirmation, with monoclonal antibodies (such as clone 1A12) showing cross-reactivity between human and chicken RHOT1 due to conserved epitopes . Mass spectrometry analysis should be employed for definitive sequence verification. For functional validation, GTPase activity can be assessed using colorimetric assays measuring phosphate release. Calcium-binding functionality can be evaluated through circular dichroism spectroscopy to detect conformational changes upon calcium addition. Researchers should note that partial recombinant constructs may exhibit different activities compared to the full-length protein. For optimal results, include appropriate positive controls (such as human RHOT1) and negative controls (heat-inactivated enzyme) in activity assays to establish baseline values and confirm specificity .

What antibodies are suitable for detecting recombinant chicken RHOT1 in experimental procedures?

Several antibodies developed against mammalian RHOT1 show cross-reactivity with chicken RHOT1 due to conserved epitopes. When selecting antibodies, consider the specific region of RHOT1 present in your recombinant construct. For partial recombinant chicken RHOT1 containing amino acids corresponding to the 483-580 region of human RHOT1, mouse monoclonal antibodies (such as clone 1A12) have demonstrated efficacy in Western blotting and immunofluorescence applications . For broader cross-species reactivity, rabbit polyclonal antibodies targeting the N-terminal region have proven effective across multiple species including zebrafish, making them potentially suitable for chicken RHOT1 detection . When performing Western blot analysis, optimize blocking conditions (typically 5% non-fat milk or BSA) and antibody dilutions (starting at 1:1000 for primary antibodies) to minimize background and enhance specific signal detection.

How conserved is chicken RHOT1 compared to mammalian orthologs?

Chicken RHOT1 exhibits significant sequence and structural conservation with mammalian orthologs, particularly within functional domains. Sequence alignment analyses reveal approximately 75-85% amino acid identity between chicken and human RHOT1, with the highest conservation observed in the GTPase domains and calcium-binding EF-hand motifs. This evolutionary conservation extends to functional mechanisms, as evidenced by the preservation of critical residues associated with GTP binding, hydrolysis, and calcium sensing. The R272 residue in human RHOT1 (corresponding to R285 in mouse), which is mutated in some Parkinson's disease patients, is also conserved in chicken RHOT1, highlighting the evolutionary importance of this amino acid position . This high degree of conservation enables researchers to extrapolate findings between species and use chicken models for studying fundamental aspects of RHOT1 biology relevant to human health and disease.

How does chicken RHOT1 contribute to mitochondrial dynamics and quality control?

Chicken RHOT1, like its mammalian counterparts, functions as a central regulator of mitochondrial dynamics and quality control pathways. When mitochondrial damage occurs, RHOT1 undergoes regulated degradation as one of the earliest events in the mitophagy cascade. This degradation is essential for detaching damaged mitochondria from the microtubule transport machinery, thereby quarantining them for subsequent autophagic clearance . Based on studies with human RHOT1, this process involves PINK1-mediated stabilization on the outer mitochondrial membrane followed by PRKN (Parkin) recruitment, which then targets RHOT1 for proteasomal degradation. This mechanism arrests mitochondrial movement, an essential prerequisite for effective mitophagy . Experimental approaches to study this process in chicken systems should include live-cell imaging with fluorescently labeled mitochondria to track immobilization dynamics, coupled with biochemical assessments of RHOT1 degradation kinetics following mitochondrial depolarization (commonly induced using CCCP treatment).

What experimental approaches are optimal for studying chicken RHOT1 mutations equivalent to human disease variants?

To investigate chicken RHOT1 mutations equivalent to human disease variants, researchers should employ a multi-tiered experimental approach. First, sequence alignment between chicken and human RHOT1 identifies conserved residues corresponding to human pathogenic mutations (e.g., R272Q, T351A, R450C, and T610A) . Site-directed mutagenesis can then generate these mutations in recombinant chicken RHOT1 constructs. For cellular studies, CRISPR/Cas9 genome editing in chicken cell lines or primary neurons allows examination of mutations in an endogenous context.

Functional assessments should include:

• Mitochondrial morphology and distribution analysis using confocal microscopy

• Mitochondrial trafficking dynamics via live-cell imaging

• Calcium binding capacity using fluorescence-based calcium sensors

• GTPase activity measurements for mutations in GTPase domains

• Interaction studies with trafficking adaptor proteins

• Mitophagy progression monitoring following mitochondrial damage

For in-depth analysis, comparing chicken cells expressing wild-type versus mutant RHOT1 under baseline and stressed conditions (e.g., CCCP treatment) provides insights into mutation-specific defects in mitochondrial clearance pathways .

How does calcium binding affect chicken RHOT1 function and what methods can assess this interaction?

Calcium binding to the EF-hand domains of chicken RHOT1 acts as a molecular switch regulating mitochondrial trafficking and distribution in response to intracellular calcium fluctuations. When calcium binds to these domains, conformational changes occur that disrupt RHOT1's interactions with kinesin adaptor proteins, resulting in mitochondrial arrest. This calcium-sensing mechanism is particularly critical in neuronal cells where mitochondrial positioning at high-energy demand sites depends on local calcium concentrations .

Several methodologies can assess this calcium-RHOT1 interaction:

• Isothermal titration calorimetry (ITC) provides direct measurement of calcium binding affinity and thermodynamic parameters

• Circular dichroism spectroscopy detects calcium-induced conformational changes

• Microscale thermophoresis offers a sensitive approach for quantifying binding constants

• Fluorescence-based calcium sensors fused to RHOT1 enable real-time monitoring in living cells

• FRET-based assays using calcium-sensitive fluorophores can detect structural rearrangements

What is the relationship between chicken RHOT1 and mitophagy, and how can this be studied experimentally?

Chicken RHOT1 functions as a critical regulator of mitophagy through mechanisms conserved with mammalian systems. Upon mitochondrial damage, RHOT1 undergoes rapid degradation, which serves as a key signal initiating the quarantine and subsequent clearance of damaged mitochondria. This process involves PINK1-mediated phosphorylation events and PRKN-dependent ubiquitination cascades that target RHOT1 for proteasomal degradation .

To study this relationship experimentally, researchers can employ several complementary approaches:

• Immunoblotting to track RHOT1 degradation kinetics following mitochondrial damage (induced by CCCP or antimycin A)

• Co-immunoprecipitation assays to detect interactions between RHOT1 and mitophagy machinery components

• Live-cell imaging with dual-labeled mitochondria and autophagosomes to visualize mitophagy progression

• CRISPR/Cas9-mediated RHOT1 knockout or knockdown to assess mitophagy dependency on RHOT1

• Flux assays measuring LC3-II accumulation in the presence of lysosomal inhibitors

Comparative studies between wild-type and mutant RHOT1 constructs can reveal how specific domains or post-translational modifications influence mitophagy rates. In particular, monitoring the co-localization of LC3 puncta with mitochondria following damage provides a quantitative measure of mitophagy progression that can be directly correlated with RHOT1 status .

How does phosphorylation regulate chicken RHOT1 activity and what kinases are involved?

Phosphorylation serves as a key post-translational regulatory mechanism for chicken RHOT1, influencing its stability, interactions, and function in mitochondrial dynamics. Based on studies with mammalian RHOT1, several phosphorylation sites have been identified, with PINK1-mediated phosphorylation being particularly critical for mitophagy initiation. PINK1 phosphorylates RHOT1 following mitochondrial damage, marking it for subsequent ubiquitination by PRKN and proteasomal degradation . Additionally, LRRK2 (Leucine-rich repeat kinase 2) has been implicated in RHOT1 regulation, with pathogenic LRRK2 mutations (such as G2019S) causing delayed RHOT1 degradation and impaired mitophagy .

To study RHOT1 phosphorylation experimentally:

• Phospho-specific antibodies can detect specific phosphorylation events

• Mass spectrometry-based phosphoproteomics provides comprehensive phosphosite mapping

• In vitro kinase assays determine direct kinase-substrate relationships

• Phosphomimetic and phosphodeficient mutants help evaluate the functional significance of specific phosphorylation events

• Proximity labeling techniques identify kinases in the RHOT1 interaction network

The interplay between different kinases creates a sophisticated regulatory network controlling RHOT1 function in response to various cellular stresses and signaling pathways .

What are the optimal buffer conditions for maintaining chicken RHOT1 stability during purification?

Establishing optimal buffer conditions is critical for maintaining the stability and functionality of recombinant chicken RHOT1 during purification. Based on protocols adapted from studies with human RHOT1, the following buffer system has proven effective: 20 mM Tris-HCl (pH 7.4-8.0), 150 mM NaCl, 1 mM DTT or 2 mM β-mercaptoethanol, and 10% glycerol as a base buffer. For constructs containing EF-hand domains, supplementation with 1-2 mM CaCl₂ enhances protein stability by promoting proper folding of these calcium-binding motifs . When purifying GTPase domain-containing constructs, addition of 1-5 mM MgCl₂ and 50-100 μM GTP or non-hydrolyzable GTP analogs helps maintain the native conformation. Protease inhibitor cocktails should be included throughout purification to prevent degradation. During concentration steps, maintain protein below 5 mg/ml to prevent aggregation, and perform buffer exchanges gradually to avoid protein precipitation. For long-term storage, flash-freezing in liquid nitrogen after adding glycerol to a final concentration of 15-20% helps preserve activity for up to 6 months at -80°C .

How can I assess the impact of mutations on chicken RHOT1 GTPase activity?

Evaluating the impact of mutations on chicken RHOT1 GTPase activity requires robust biochemical assays with appropriate controls. The malachite green assay presents a reliable colorimetric method for measuring inorganic phosphate release from GTP hydrolysis. For this approach, purified wild-type and mutant RHOT1 proteins (1-5 μM) are incubated with GTP (typically 0.5-1 mM) in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM MgCl₂ at 37°C. Aliquots removed at different time points and mixed with malachite green reagent enable quantification of phosphate release rates, allowing direct comparison between wild-type and mutant proteins .

Alternative approaches include:

• HPLC-based methods to directly measure GTP to GDP conversion

• Fluorescent GTP analogs (like BODIPY-GTP) to monitor binding and hydrolysis kinetically

• Radiometric assays using [γ-³²P]GTP for high sensitivity measurements

• Isothermal titration calorimetry to determine thermodynamic parameters of GTP binding

When analyzing GTPase-deficient mutations (such as those equivalent to human R272Q or R450C), include both positive controls (wild-type protein) and negative controls (heat-denatured protein or known GTPase-dead mutants) to establish the dynamic range of your assay system .

What are the most reliable methods for studying chicken RHOT1-mediated mitochondrial trafficking?

Studying chicken RHOT1-mediated mitochondrial trafficking requires techniques that capture the dynamic nature of mitochondrial movement while providing quantitative data. Live-cell imaging using fluorescently-labeled mitochondria (typically with MitoTracker dyes or mitochondria-targeted fluorescent proteins) represents the gold standard approach. For primary chicken neurons or established cell lines, time-lapse confocal microscopy with 5-10 second intervals over 5-10 minutes provides sufficient temporal resolution to track individual mitochondria .

Recommended analytical parameters include:

For manipulating RHOT1 function, combine imaging with:

• Expression of wild-type vs. mutant RHOT1 constructs

• RHOT1 knockdown/knockout using siRNA or CRISPR/Cas9

• Chemical inhibitors of mitochondrial function (CCCP) to assess damage responses

• Calcium ionophores to evaluate calcium-dependent trafficking regulation

How can I develop a chicken cell model to study RHOT1 mutations?

Developing chicken cell models for studying RHOT1 mutations requires strategic selection of appropriate cell types and genetic modification techniques. For basic studies, chicken fibroblast lines (such as DF-1) provide a readily manipulable system amenable to standard transfection methods. For neuron-specific investigations, primary embryonic neuronal cultures offer a more physiologically relevant context, though they present greater technical challenges.

For genetic modification, several approaches are viable:

• CRISPR/Cas9 genome editing: Design guide RNAs targeting conserved regions of chicken RHOT1, along with appropriate repair templates containing your mutation of interest. Optimize transfection conditions for your specific cell type (typically electroporation for primary neurons, lipid-based transfection for established lines).

• Lentiviral expression systems: For stable expression of wild-type or mutant RHOT1, lentiviral vectors under neuronal-specific promoters (like synapsin) ensure appropriate expression levels and cell-type specificity.

• Inducible expression systems: Tet-On/Off systems allow temporal control of mutant RHOT1 expression, facilitating the study of acute versus chronic effects.

For phenotypic analysis, implement multiparameter assessments including mitochondrial morphology, distribution, membrane potential, and trafficking dynamics. Complementary biochemical assays should measure mitophagy flux (LC3-II levels with and without lysosomal inhibitors) and mitochondrial function (oxygen consumption, ATP production) .

What experimental approaches can measure the effects of chicken RHOT1 on mitochondrial bioenergetics?

Assessing the impact of chicken RHOT1 on mitochondrial bioenergetics requires a multi-faceted approach combining real-time metabolic measurements with endpoint biochemical assays. Seahorse XF analyzers provide a powerful platform for measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in living cells, enabling quantification of key parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity. When designing these experiments, compare wild-type cells to those expressing mutant RHOT1 constructs or RHOT1 knockdown/knockout models .

Complementary approaches include:

• High-resolution respirometry (Oroboros O2k) for detailed analysis of individual respiratory complexes

• ATP bioluminescence assays to directly measure cellular ATP levels

• NAD⁺/NADH ratio determination as an indicator of cellular redox state

• Mitochondrial membrane potential assessment using potential-sensitive dyes (TMRM, JC-1)

• Live-cell imaging with genetically-encoded ATP or reactive oxygen species sensors

For metabolomic profiling, targeted LC-MS/MS analysis focusing on TCA cycle intermediates, amino acids, and acylcarnitines can reveal specific metabolic alterations associated with RHOT1 dysfunction. As demonstrated in studies of human RHOT1 mutations, these approaches can identify significant changes in cellular energy metabolism that contribute to pathological states .

What role might chicken RHOT1 play in mitochondrial adaptation to metabolic stress?

Chicken RHOT1 likely serves as a crucial mediator in mitochondrial adaptation to metabolic stress, functioning as both a sensor and effector in stress response pathways. Under metabolic stress conditions such as nutrient deprivation or increased energy demand, RHOT1-mediated mitochondrial trafficking ensures proper distribution of mitochondria to high-energy-demanding regions. Additionally, RHOT1's calcium-sensing ability enables rapid responses to calcium fluctuations that often accompany metabolic perturbations .

Experimental approaches to investigate this role include:

• Subjecting chicken cells with normal or altered RHOT1 expression to various metabolic stressors (glucose deprivation, fatty acid overload, or electron transport chain inhibitors)

• Monitoring mitochondrial redistribution patterns during stress using live-cell imaging

• Assessing mitochondrial network dynamics through fusion/fission balance quantification

• Measuring adaptive responses including mitochondrial biogenesis and mitophagy flux

• Evaluating metabolic flexibility through substrate utilization patterns under stress conditions

Preliminary evidence from mammalian systems suggests RHOT1's involvement in metabolic adaptation extends beyond trafficking to include roles in mitochondrial quality control and communication with other cellular compartments. Investigating these aspects in chicken models could reveal conserved mechanisms of cellular resilience to metabolic challenges .

What are the emerging technologies that will advance chicken RHOT1 research?

Emerging technologies are poised to revolutionize chicken RHOT1 research, enabling unprecedented insights into its structure, dynamics, and function. Cryo-electron microscopy advancements now allow visualization of membrane proteins in near-native states, offering potential for resolving the full-length chicken RHOT1 structure, including its transmembrane domain and interactions with binding partners. This structural information would significantly enhance our understanding of how disease-associated mutations affect protein function .

Single-molecule imaging techniques, including super-resolution approaches like STORM and PALM, enable tracking of individual RHOT1 molecules in living cells, providing direct visualization of its dynamics during mitochondrial movement and mitophagy. These techniques can be combined with optogenetic tools to manipulate RHOT1 function with precise spatiotemporal control .

Genome editing technologies continue to evolve, with base editing and prime editing offering higher precision than traditional CRISPR/Cas9 for introducing specific point mutations modeling disease variants. These approaches minimize off-target effects and unintended genomic alterations .

For functional studies, advances in mitochondrial-targeted biosensors enable real-time monitoring of various parameters including calcium levels, membrane potential, and ROS production specifically within the mitochondrial microenvironment where RHOT1 functions. Combining these biosensors with high-content imaging platforms facilitates multi-parametric assessment of RHOT1's impact on mitochondrial physiology .

How might chicken RHOT1 research translate to therapeutic strategies for human diseases?

Research on chicken RHOT1 contributes valuable insights that could inform novel therapeutic strategies for human mitochondrial diseases and neurodegenerative disorders, particularly Parkinson's disease (PD). The high degree of evolutionary conservation between chicken and human RHOT1 makes findings potentially translatable to human health applications .

Several promising therapeutic directions emerge from this research:

• Targeted modulation of RHOT1 degradation: Evidence indicates that partial knockdown of RHOT1 in LRRK2 G2019S mutant neurons promotes mitochondrial arrest and mitophagy, enhancing neuronal survival. This suggests that calibrated reduction of RHOT1 levels or activity could benefit patients with specific genetic forms of PD .

• Enhancing mitophagy: Compounds that facilitate RHOT1 degradation following mitochondrial damage could accelerate mitophagy and prevent the accumulation of dysfunctional mitochondria observed in neurodegenerative conditions .

• RHOT1 as a biomarker: The consistent finding of delayed RHOT1 degradation (RHOT1 retention) across multiple forms of PD suggests its potential as an early biomarker for disease diagnosis and monitoring treatment efficacy .

• Calcium homeostasis modulation: Targeting the calcium-sensing function of RHOT1 might help normalize mitochondrial distribution and function in diseases characterized by calcium dysregulation .