Recombinant Mouse Centrosomal protein KIAA1731 homolog (Kiaa1731), partial

What is KIAA1731 and what are its main cellular functions?

KIAA1731 is a centrosomal protein that plays essential roles in maintaining centrosome structure and function. The protein was initially identified as a candidate centrosomal component through proteomic analysis . Functionally, KIAA1731 appears critical for centriole biogenesis or stability, as evidenced by the gradual loss of centrioles in cells depleted of this protein .

The protein contains an ALMS motif at its C-terminus, similar to that found in C10orf90, suggesting shared functional elements between these proteins . Additionally, KIAA1731 contains an N-terminal region with similarity to Ddc8, a protein highly expressed in testis that localizes to the tails of elongated spermatids and spermatozoa . This structural organization points to multiple potential functions in cellular processes beyond basic centriole formation.

Analysis of KIAA1731-depleted cells shows that without this protein, cells exhibit mitotic abnormalities including monopolar spindles and asymmetric distribution of centrosomal components, highlighting its importance in proper cell division .

What experimental approaches are most effective for studying mouse KIAA1731 localization?

Immunofluorescence microscopy represents the gold standard for studying KIAA1731 localization. Based on studies with human KIAA1731, tagged constructs colocalizing with centriolar acetylated tubulin provide excellent visualization of the protein's distribution . For mouse KIAA1731 homolog, researchers should consider:

• Generating epitope-tagged constructs (Myc-tagged or GFP-tagged) of mouse KIAA1731 for transfection studies

• Co-staining with established centrosomal markers like γ-tubulin and acetylated tubulin

• Super-resolution microscopy for precise subcellular localization within centrosomal structures

• Immunoelectron microscopy for ultrastructural localization

When studying localization, it's important to note that human KIAA1731 shows distinct localization patterns compared to related proteins like ALMS1. While ALMS1 targets the proximal ends of centrioles, KIAA1731 appears to distribute along centriolar structures more broadly, similar to Drosophila Ana1 .

What phenotypic effects are observed following KIAA1731 depletion in cellular models?

KIAA1731 depletion produces several distinct and progressive cellular phenotypes that demonstrate its critical role in centrosome maintenance and function:

• Gradual reduction in centriole numbers over time, consistent with defects in centriole biogenesis rather than immediate structural collapse

• At 48 hours post-depletion, cells containing a single centriole appear at approximately twice the frequency of cells completely lacking centrioles

• By 96 hours post-depletion, cells completely lacking centrioles become more prevalent than those with a single centriole

• Mitotic cells exhibit monopolar spindles with only one focus of acetylated tubulin/γ-tubulin

• Abnormal chromosome arrangements during metaphase, suggesting failed bipolar spindle assembly

• Some mitotic cells display highly asymmetric distribution of γ-tubulin between spindle poles, similar to cells depleted of CPAP (another protein required for centriole formation)

These phenotypes indicate that KIAA1731 is not merely a structural component of centrosomes but plays an active role in the biogenesis or maintenance of centrioles throughout the cell cycle.

How does mouse KIAA1731 homolog compare structurally to human KIAA1731?

While the search results don't provide direct comparative data between mouse and human KIAA1731, general principles of homologous proteins suggest several important considerations for researchers:

When working with recombinant mouse KIAA1731, researchers should validate that structural features critical to centrosomal localization and function are preserved compared to the human ortholog.

What techniques are recommended for validating KIAA1731 knockdown efficiency?

Based on approaches used in human KIAA1731 studies, several techniques are recommended for validating knockdown efficiency:

• Quantitative PCR (qPCR) using predesigned Taqman Gene Expression Assays, with HPRT as a reference gene for normalization

• Semi-quantitative RT-PCR with gene-specific primers, visualized on agarose gels (can provide a quicker initial assessment before quantification)

• Immunoblot analysis of cell lysates:

  • Prepare lysates in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, with protease inhibitors

  • Clear lysates by centrifugation (13,000 rpm, 10 min, 4°C)

  • Separate proteins by SDS-PAGE and transfer to appropriate membranes

  • Probe with anti-KIAA1731 antibodies

• Immunofluorescence microscopy to assess protein depletion at the centrosome, which provides spatial information about knockdown effects

For siRNA-based approaches, researchers should design multiple siRNAs targeting different regions of the transcript to minimize off-target effects, similar to the approach used in human studies (e.g., KIAA1731_4333 and KIAA1731_03) .

What is the functional significance of the ALMS motif in KIAA1731?

The ALMS motif represents a key functional domain of KIAA1731 with significant implications for centrosomal targeting and function. This motif was initially identified in ALMS1, a protein implicated in Alström syndrome (a ciliopathy), and later found in both KIAA1731 and C10orf90 .

The ALMS motif appears to be the only element within KIAA1731 that bears significant similarity to other human proteins, suggesting its evolutionary conservation reflects important functional constraints . Current evidence indicates this motif may contribute to:

• Centrosomal targeting: Tagged constructs containing KIAA1731 localize to centriolar structures, suggesting the ALMS motif may participate in this localization

• Protein-protein interactions: The presence of similar motifs in functionally related proteins (ALMS1, C10orf90, KIAA1731) suggests this domain may mediate interactions with common binding partners at the centrosome

• Structural integrity: The phenotypes observed upon KIAA1731 depletion suggest this protein, through its ALMS motif, may contribute to maintaining centriole structural stability

Experimental approaches to further elucidate ALMS motif function could include domain deletion analyses, site-directed mutagenesis of conserved residues, and identification of interacting proteins specific to this region.

How does KIAA1731 contribute to centriole biogenesis at the molecular level?

KIAA1731's role in centriole biogenesis appears to be fundamental, with depletion studies providing strong evidence for its necessity in this process. At the molecular level, several mechanisms are suggested by current research:

• Temporal dynamics: KIAA1731 depletion leads to a pattern of centriole loss consistent with a role in centriole formation rather than maintenance alone—cells progressively lose centrioles through successive divisions, suggesting failure of the duplication process

• Similarity to Ana1: KIAA1731 shares sequence similarity with Drosophila Ana1, a protein implicated in centriole duplication. This evolutionary relationship suggests conserved functions in the centriole assembly pathway

• Structural localization: Unlike ALMS1, which localizes specifically to proximal ends of centrioles, KIAA1731 appears to localize along the length of centrioles, similar to Ana1 in Drosophila. This distribution pattern may reflect a role in centriole elongation or stability rather than initiation

• Cell cycle relevance: The centriole loss phenotype includes mitotic defects with monopolar spindles and chromosome segregation abnormalities, indicating KIAA1731's role intersects with cell cycle progression mechanisms

While KIAA1731 is likely involved in the centriole duplication pathway, its precise position in the hierarchy of centriole assembly factors requires further investigation. Recent studies of Ana1 suggest it may not be an upstream regulator of centriole duplication, implying KIAA1731 may similarly function as a structural or stabilizing component rather than an initiating factor .

What evolutionary relationships exist between KIAA1731 and its homologs across species?

The evolutionary relationships of KIAA1731 reveal important insights about its conserved functions:

This pattern of conservation—maintained functional relationships despite substantial sequence divergence—suggests strong selective pressure on specific functional domains rather than the entire protein sequence. This evolutionary pattern is often observed in proteins that serve as structural scaffolds where specific interaction interfaces remain conserved while intervening regions diverge.

The conservation between mouse and human KIAA1731 homologs would be expected to be substantially higher than between human and Drosophila proteins, reflecting their closer evolutionary relationship.

What methodological approaches should be used to analyze potential KIAA1731 involvement in ciliopathies?

Given KIAA1731's likely role in the formation/stability of basal bodies and the cilium assembly process, several methodological approaches are recommended for investigating its potential involvement in ciliopathies:

• Genetic screening of ciliopathy patients:

  • Targeted sequencing of KIAA1731 in patients with ciliopathies of unknown genetic cause

  • Whole exome/genome sequencing with focused analysis of KIAA1731 variants

  • Assessment of copy number variations affecting the KIAA1731 locus

• Functional assays in cellular models:

  • Cilium formation assays following serum starvation in KIAA1731-depleted cells

  • Analysis of cilium length, structure, and composition using immunofluorescence microscopy

  • Live imaging of cilium formation in cells expressing fluorescently-tagged KIAA1731

• Mouse model studies:

  • Generation of KIAA1731 knockout or knockin mice carrying human disease mutations

  • Phenotypic analysis focusing on classic ciliopathy features (retinal degeneration, polydactyly, kidney cysts, etc.)

  • Tissue-specific conditional knockouts to assess organ-specific requirements

• Protein interaction studies:

  • Identification of KIAA1731 binding partners among known ciliopathy proteins

  • Analysis of how disease-associated mutations affect these interactions

  • Investigation of KIAA1731's relationship with intraflagellar transport machinery

The phenotypic overlap between KIAA1731 depletion (affecting centriole formation) and C10orf90 depletion (affecting cilium formation) suggests these ALMS motif-containing proteins may participate in related pathways relevant to ciliopathies .

What are the key considerations for designing experiments with recombinant partial mouse KIAA1731?

When working with recombinant partial mouse KIAA1731, researchers should consider several critical experimental design factors:

• Domain selection and boundaries:

  • Ensure the partial construct contains complete functional domains (e.g., ALMS motif and/or Ddc8-like region)

  • Consider secondary structure predictions to avoid disrupting folded domains

  • Include flexible linkers if multiple domains are being combined

• Expression system considerations:

  • Mammalian expression systems may provide appropriate post-translational modifications

  • Bacterial systems may be suitable for structural studies of individual domains

  • Codon optimization for the selected expression system

• Fusion tag selection:

  • N-terminal vs. C-terminal tags may differentially affect function

  • Tag removal options (protease cleavage sites)

  • Consider tag effects on localization (evidence suggests N-terminal tags on KIAA1731 preserve centrosomal localization)

• Functional validation:

  • Rescue experiments in KIAA1731-depleted cells to confirm functionality of recombinant constructs

  • Localization studies comparing endogenous and recombinant protein distribution

  • Protein-protein interaction studies to confirm binding partner engagement

• Potential artifacts:

  • Overexpression of control proteins (e.g., eGFP) can affect subcellular distribution of centrosomal proteins like PCM1, potentially complicating rescue experiments

  • Expression level control is critical, as excessive protein may form aggregates or mislocalize

The experimental approach should be tailored to the specific research question, with careful attention to how the recombinant construct design may impact protein function.

What RNAi approaches are most effective for studying KIAA1731 function?

Based on published methodologies for human KIAA1731, the following RNAi approaches can be adapted for mouse studies:

• siRNA design strategies:

  • Target multiple regions of the transcript (e.g., KIAA1731_4333 and KIAA1731_03 sequences used in human studies)

  • Design species-specific siRNAs accounting for sequence differences between mouse and human

  • Include appropriate negative controls (e.g., AllStars Negative Control siRNA)

• Transfection methods:

  • Lipid-based transfection (e.g., Lipofectamine 2000) for most cell types

  • Nucleofection for difficult-to-transfect cell types

  • Viral vector delivery for long-term knockdown or in vivo studies

• Knockdown validation:

  • qPCR with mouse-specific primers and probes, using HPRT as a reference gene

  • Semi-quantitative RT-PCR with endpoint analysis

  • Immunoblotting with antibodies specific to mouse KIAA1731

• Phenotypic analysis timeline:

  • Assess phenotypes at multiple timepoints (48h, 72h, 96h) to capture progression of centriole loss

  • For cilium formation studies, induce ciliation by serum starvation following siRNA treatment

• Controls and rescue experiments:

  • Include siRNA-resistant rescue constructs to confirm specificity

  • Be aware that overexpression of control proteins can sometimes affect centrosomal protein distribution, complicating interpretation

The progressive nature of the centriole loss phenotype means experimental timing is crucial—early timepoints may show subtle effects that become more pronounced with continued cell division.

What are the appropriate centrosomal markers for co-localization studies with mouse KIAA1731?

For comprehensive analysis of KIAA1731 localization within centrosomal structures, the following markers are recommended:

• Centriolar markers:

  • Acetylated α-tubulin: Labels the centriolar microtubules and is effective for visualizing centriole numbers and structure

  • Centrin: Marks the distal end of centrioles

  • SAS-6: Labels the cartwheel structure during early centriole formation

• Pericentriolar material (PCM) markers:

  • γ-tubulin: Core component of the PCM and useful for assessing centrosome integrity

  • Pericentrin: Scaffolding protein of the PCM

  • PCM1: Satellite protein that can be affected by KIAA1731 depletion

• Proximal end markers:

  • C-Nap1: Marks the proximal end of centrioles and is involved in centrosome cohesion

  • CEP135: Proximal end marker that connects to the cartwheel structure

• Cell cycle specific markers:

  • Centrobin: Preferentially associates with daughter centrioles

  • Plk4: Master regulator of centriole duplication

  • CPAP: Required for centriole elongation

When designing co-localization experiments, super-resolution microscopy techniques (e.g., SIM, STED, or STORM) can provide significantly improved resolution of centrosomal structures compared to conventional confocal microscopy.

How can researchers effectively analyze centriole biogenesis defects in KIAA1731-deficient models?

Based on methodologies used in human KIAA1731 studies, the following approaches are recommended for analyzing centriole biogenesis defects:

• Quantitative analysis of centriole numbers:

  • Immunofluorescence microscopy with centriole markers (acetylated tubulin)

  • Count centrioles in large numbers of cells (>100 per condition)

  • Categorize cells by centriole number (0, 1, 2, >2)

  • Track changes across multiple timepoints post-knockdown (48h, 72h, 96h)

• Cell cycle analysis:

  • Co-stain for cell cycle markers to determine stage-specific effects

  • Analyze mitotic cells for spindle pole defects

  • Assess chromosome alignment/segregation abnormalities

• Live cell imaging:

  • Track centriole behavior through cell cycles using fluorescently-tagged centriolar proteins

  • Monitor cell division outcomes following centriole loss

• Transmission electron microscopy:

  • Ultrastructural analysis of remaining centrioles to assess structural integrity

  • Examination of abnormal centriole intermediates

• Data presentation:

  • Quantify the percentage of cells in each centriole number category at each timepoint

  • Create time-course graphs showing the progressive loss of centrioles

  • Document representative images of the observed phenotypes

Example data table for centriole quantification based on the human KIAA1731 depletion model:

Note: Values estimated based on trends described in the literature . Exact percentages would vary by cell type and knockdown efficiency.

What approaches can be used to investigate potential interaction partners of mouse KIAA1731?

Identification and characterization of KIAA1731 interaction partners are critical for understanding its molecular functions. Several complementary approaches are recommended:

• Immunoprecipitation-based methods:

  • Co-immunoprecipitation of endogenous proteins

  • GFP-Trap or other tag-based pulldowns of recombinant KIAA1731

  • Crosslinking immunoprecipitation for transient or weak interactions

• Proximity labeling approaches:

  • BioID fusion to KIAA1731 to identify proximal proteins in living cells

  • APEX2 fusion for rapid proximity labeling with electron microscopy compatibility

• Domain-specific interaction mapping:

  • Yeast two-hybrid screening with specific domains (e.g., ALMS motif, Ddc8-like region)

  • Peptide array analysis to identify short linear interacting motifs

• Candidate-based approaches:

  • Focus on known centrosomal proteins, particularly those involved in centriole biogenesis

  • Investigate interactions with Ana1-interacting proteins identified in Drosophila

  • Test interactions with ALMS1 and C10orf90, which share the ALMS motif

• Analysis of interaction dependencies:

  • Assess how mutations in key domains affect interaction profiles

  • Determine cell cycle regulation of interactions

  • Investigate how centrosome disrupting drugs affect KIAA1731 interactions

When identifying novel interaction partners, validation through multiple techniques is essential, as centrosomal proteins can produce false positives in some interaction assays due to their insolubility or participation in large complexes.

How might KIAA1731 mutations contribute to ciliopathies and other centrosome-related disorders?

KIAA1731's critical role in centriole formation/stability suggests several potential mechanisms by which mutations could contribute to disease:

• Ciliopathy mechanisms:

  • Disruption of basal body formation: Since basal bodies are derived from centrioles, KIAA1731 dysfunction could impair the foundation for cilium assembly

  • Secondary effects on cilium formation: Even if basal bodies form, their structural abnormalities might prevent proper ciliary axoneme extension

  • Tissue-specific requirements: Different tissues may have varying thresholds for KIAA1731 function, explaining the tissue-specific manifestations of ciliopathies

• Potential disease associations:

  • Primary ciliary dyskinesia: Defects in motile cilia function

  • Nephronophthisis: Kidney cysts and dysfunction

  • Retinal degeneration: Photoreceptor death due to defective connecting cilia

  • Polydactyly and skeletal abnormalities: Disrupted Hedgehog signaling during development

• Cancer implications:

  • Centrosome amplification: While complete loss of KIAA1731 leads to centriole loss, hypomorphic mutations might cause centrosome instability leading to numerical abnormalities

  • Mitotic spindle defects: Abnormal chromosome segregation due to monopolar or asymmetric spindles

  • Cell cycle checkpoint activation: Prolonged mitosis or mitotic catastrophe

The molecular evidence suggesting KIAA1731 mutations could represent novel causes of human ciliopathies warrants dedicated genetic screening of patient cohorts with undiagnosed ciliopathies.

What strategies could be employed to develop therapeutic approaches targeting KIAA1731-related disorders?

Therapeutic development for KIAA1731-related disorders would depend on the specific disease mechanism, but several approaches could be considered:

• Gene therapy approaches:

  • AAV-mediated delivery of functional KIAA1731 to affected tissues

  • CRISPR-based correction of disease-causing mutations

  • Antisense oligonucleotides to promote correct splicing if mutations affect splicing

• Small molecule strategies:

  • Compounds that stabilize partially functional KIAA1731 mutant proteins

  • Drugs targeting downstream pathways affected by KIAA1731 dysfunction

  • Chaperone-based therapies to improve folding of destabilized mutants

• Cell-based therapies:

  • Stem cell therapies for tissue regeneration in degenerative ciliopathies

  • Ex vivo genetic correction followed by autologous transplantation

• Phenotypic screening considerations:

  • High-content imaging assays measuring centriole number/morphology

  • Cilium formation assays in patient-derived cells

  • Organoid models to assess tissue-specific ciliary functions

• Model systems for therapeutic testing:

  • Patient-derived iPSCs and differentiated derivatives

  • CRISPR-engineered cell lines carrying specific patient mutations

  • Mouse models with corresponding mutations in the Kiaa1731 gene

Since many ciliopathies affect multiple organ systems, therapeutic approaches may need to be tailored to specific tissues, potentially requiring diverse delivery strategies depending on the affected organs.