Recombinant Chicken TBCC domain-containing protein 1 (TBCCD1), partial

What is TBCCD1 and what cellular functions does it perform?

TBCCD1 (TBCC domain-containing protein 1) is a protein related to tubulin cofactor C that plays a crucial role in centrosome positioning. It localizes primarily at the centrosome and at the spindle midzone, midbody, and basal bodies of primary and motile cilia . Unlike its relative TBCC, TBCCD1 lacks the conserved arginine residue crucial for GAP activity towards tubulin, suggesting different functional properties .

Research using human TBCCD1 has demonstrated that it acts as a key regulator of centrosome-nucleus association. When TBCCD1 is depleted, the centrosome becomes dissociated from the nucleus and often relocates to the cell periphery, resulting in Golgi apparatus disorganization . While TBCCD1 is critical for proper centrosome positioning, it does not appear to affect the microtubule-nucleating activity of the centrosome .

What is the difference between full-length and partial recombinant TBCCD1?

Partial recombinant TBCCD1 contains only a segment of the complete protein sequence, typically including functional domains of interest. The partial chicken TBCCD1 likely contains the TBCC domain, which is conserved across species and related to the domain found in tubulin cofactor C .

The full-length protein would contain all domains, including the CARP domain (found in Cyclase-associated proteins) that is present in TBCC, RP2, and TBCCD1 . Research with human TBCCD1 has shown that the amino-terminal domain (aa 1-328) is involved in centrosome targeting . When designing experiments, researchers should consider whether their specific research questions require the complete protein or if the partial recombinant containing key functional domains is sufficient.

How is TBCCD1 evolutionarily conserved across species?

TBCCD1 is conserved throughout the phylogenetic tree, suggesting fundamental roles in cellular function . The human and chicken variants share significant homology, particularly in the conserved TBCC and CARP domains. Evolutionary conservation analysis indicates that TBCCD1 is related to TBCC, which together with TBCD acts as a β-tubulin GTPase-activating protein (GAP) .

TBCCD1 is also related to RP2, which overlaps functionally with TBCC and seems to participate in tubulin quality control at the basal body of flagella in organisms such as Trypanosoma . This evolutionary conservation makes chicken TBCCD1 a valuable model for comparative studies of centrosome function across species.

What are the optimal storage and handling conditions for recombinant chicken TBCCD1?

For optimal stability and activity, recombinant chicken TBCCD1 should be stored according to the following guidelines:

• Lyophilized form can be stored for up to 12 months at -20°C/-80°C

• Liquid form has a shelf life of approximately 6 months at -20°C/-80°C

• After reconstitution, working aliquots should be stored at 4°C for no more than one week

• Repeated freezing and thawing should be avoided to maintain protein integrity

For reconstitution, it is recommended to:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation)

• Create aliquots for long-term storage at -20°C/-80°C

What experimental approaches can be used to study TBCCD1's role in centrosome positioning?

To investigate TBCCD1's role in centrosome positioning, researchers can employ several methodological approaches:

• RNAi-mediated knockdown: Following the approach used with human TBCCD1, researchers can transfect cells with siRNAs directed at chicken TBCCD1. For effective knockdown, a pool of four siRNAs can be used at approximately 100 nM concentration with a suitable transfection reagent like Oligofectamine .

• Immunofluorescence analysis: To visualize centrosome positioning, researchers can use antibodies against centrosomal markers (like γ-tubulin) in combination with DAPI staining for the nucleus. The distance between the centrosome and nucleus can then be measured to quantify positioning defects .

• Microtubule regrowth assays: To assess whether TBCCD1 affects centrosomal microtubule nucleation, microtubules can be depolymerized with nocodazole (30 μM for 40 minutes) and then allowed to repolymerize after washout .

• Live cell imaging: For dynamic studies of centrosome positioning, cells expressing fluorescently tagged centrosomal proteins can be monitored in real-time using time-lapse microscopy .

How can researchers validate the specificity and efficiency of TBCCD1 knockdown?

For rigorous validation of TBCCD1 knockdown experiments, researchers should implement multiple confirmation methods:

• RT-PCR analysis: Quantify TBCCD1 mRNA levels in control versus knockdown samples to confirm reduced transcription .

• Western blotting: Assess protein levels using specific anti-TBCCD1 antibodies to confirm reduced expression at the protein level .

• Immunofluorescence: Perform immunostaining to visualize decreased TBCCD1 at the centrosome and cytoplasm in knockdown cells .

• Phenotypic rescue: To confirm specificity, perform rescue experiments by expressing an siRNA-resistant version of TBCCD1 to see if it reverses the observed phenotypes .

• Multiple siRNA controls: Use both negative control siRNAs (non-targeting) and multiple different TBCCD1-targeting siRNAs to rule out off-target effects .

How can recombinant TBCCD1 be used to study centrosome-nucleus association mechanisms?

Recombinant TBCCD1 can serve as a valuable tool for investigating the molecular mechanisms of centrosome-nucleus association through several advanced approaches:

• In vitro binding assays: Use purified recombinant TBCCD1 to identify direct binding partners from nuclear envelope or centrosomal extracts, which could reveal the molecular linkers involved in this association.

• Domain mapping studies: Compare the effects of full-length versus partial TBCCD1 constructs to identify which domains are essential for centrosome-nucleus association. Human TBCCD1 studies have shown that the amino-terminal domain (aa 1-328) is involved in centrosome targeting .

• Proximity labeling: Fuse TBCCD1 with proximity labeling enzymes (BioID or APEX) to identify proteins in close proximity to TBCCD1 in living cells, potentially revealing the interaction network.

• Forced localization experiments: Tether TBCCD1 to specific cellular locations using optogenetic or chemically inducible systems to determine whether TBCCD1 positioning is sufficient to drive centrosome repositioning.

• Comparative studies: Compare the function of chicken TBCCD1 with human TBCCD1 to identify evolutionarily conserved mechanisms of centrosome positioning.

What techniques can be employed to investigate TBCCD1's relationship with Golgi apparatus organization?

TBCCD1 depletion is known to cause Golgi apparatus disorganization . Researchers can investigate this relationship using:

• 3D confocal microscopy: Visualize the spatial relationship between TBCCD1, centrosomes, and Golgi apparatus using multilabel immunofluorescence and 3D reconstruction techniques.

• Live imaging of Golgi dynamics: Use fluorescently labeled Golgi markers in combination with TBCCD1 manipulation to track real-time changes in Golgi positioning and morphology.

• Electron microscopy: Examine ultrastructural changes in Golgi architecture following TBCCD1 depletion or overexpression.

• Drug-induced Golgi disassembly and reassembly: Compare Golgi reassembly kinetics in control versus TBCCD1-depleted cells following treatment with drugs like Brefeldin A that reversibly disrupt the Golgi.

• Quantitative image analysis: Develop algorithms to measure Golgi compactness, fragmentation, and distance from the centrosome to objectively quantify the effects of TBCCD1 manipulation.

How does TBCCD1 function in primary cilia formation and what methodologies can assess this role?

TBCCD1 has been implicated in primary cilia assembly, with knockdown resulting in reduced ciliogenesis efficiency . To investigate this function:

• Serum starvation protocols: Induce primary cilia formation by serum starvation in control and TBCCD1-depleted cells, then quantify the percentage of ciliated cells using immunofluorescence for ciliary markers .

• Super-resolution microscopy: Examine the precise localization of TBCCD1 at basal bodies and detect potential structural abnormalities in TBCCD1-depleted cells.

• Ciliary protein trafficking assays: Assess whether TBCCD1 affects the transport of proteins to the cilium using fluorescently tagged ciliary proteins.

• Ciliary signaling pathway analysis: Investigate whether TBCCD1 depletion affects cilium-dependent signaling pathways such as Hedgehog signaling.

• Comparative analysis across cell types: Compare the requirement for TBCCD1 in ciliogenesis across different cell types, including those that form motile versus primary cilia.

How can researchers distinguish between direct and indirect effects of TBCCD1 manipulation?

Distinguishing direct from indirect effects of TBCCD1 manipulation requires careful experimental design and controls:

• Acute versus chronic depletion: Compare phenotypes resulting from acute depletion (using degron tags or rapid protein inactivation) versus long-term knockdown to separate primary from secondary effects.

• Structure-function analysis: Create and test point mutations in specific domains to determine which protein interactions or activities are essential for each observed phenotype.

• Temporal analysis: Track the timeline of phenotypic changes after TBCCD1 depletion to establish the sequence of events and identify likely primary effects.

• Parallel pathway analysis: Simultaneously monitor multiple cellular processes (centrosome positioning, Golgi organization, cell cycle progression) to develop a comprehensive model of cause-and-effect relationships.

• Mathematical modeling: Develop quantitative models incorporating known parameters of microtubule dynamics and organelle positioning to predict which effects could be direct consequences of TBCCD1 loss.

What controls should be included when studying chicken TBCCD1 in heterologous systems?

When studying chicken TBCCD1 in non-avian cell systems, the following controls are essential:

How can researchers interpret contradictory results in TBCCD1 functional studies across different cell types?

When faced with contradictory results across different experimental systems:

• Cell type-specific expression analysis: Quantify endogenous TBCCD1 levels across cell types to determine whether expression differences might explain functional variations.

• Interactome comparison: Identify TBCCD1 binding partners in different cell types to determine whether cell type-specific interactions might alter TBCCD1 function.

• Post-translational modification analysis: Investigate whether TBCCD1 undergoes different post-translational modifications in various cell types, potentially explaining functional differences.

• Redundancy mechanisms: Assess the expression of functionally related proteins that might compensate for TBCCD1 loss in some cell types but not others.

• Experimental condition standardization: Carefully control cell density, passage number, and culture conditions, as TBCCD1 function in processes like ciliation is known to be affected by confluence .

What are promising approaches to identify TBCCD1 interaction partners and regulatory mechanisms?

To advance understanding of TBCCD1 regulatory networks:

• Proximity-dependent labeling: Apply BioID or APEX2 proximity labeling to map the TBCCD1 interactome at different cellular locations (centrosome, basal body, cytoplasm).

• Phosphoproteomics: Identify potential phosphorylation sites on TBCCD1 and the kinases/phosphatases that might regulate its activity through post-translational modifications.

• CRISPR screening: Perform genome-wide CRISPR screens to identify genes that, when knocked out, either enhance or suppress TBCCD1 depletion phenotypes.

• Structural studies: Obtain crystal or cryo-EM structures of TBCCD1 alone and in complex with binding partners to understand molecular mechanisms of interaction.

• Single-molecule imaging: Apply super-resolution techniques to track individual TBCCD1 molecules in living cells, revealing dynamic associations and potential regulatory mechanisms.

How might TBCCD1 research contribute to understanding human diseases related to centrosome dysfunction?

TBCCD1 research has potential implications for understanding several disease states:

• Ciliopathies: Since TBCCD1 affects cilia formation , studying its function could provide insights into ciliopathies—disorders resulting from dysfunctional cilia.

• Cell migration disorders: TBCCD1's role in cell migration suggests potential relevance to wound healing defects and developmental disorders involving cell migration.

• Neurological disorders: Given TBCCD1's presence in brain tissue and the importance of centrosome function in neuronal development, its dysfunction might contribute to neurodevelopmental disorders.

• Cancer research: Centrosome abnormalities are common in cancer cells; understanding TBCCD1's role in maintaining proper centrosome positioning could illuminate mechanisms of genomic instability in cancer.

• Comparative pathology: Studying the chicken TBCCD1 variant may reveal evolutionarily conserved mechanisms that can be translated to human disease models.