TTLL9 Antibody

What is the primary function of TTLL9 in ciliated cells?

TTLL9 functions as a tubulin-polyglutamylating enzyme belonging to the tubulin tyrosine ligase-like protein (TTLL) family. It forms a complex with FAP234 (Flagella-Associated Protein 234) that catalyzes the polyglutamylation of axonemal tubulin. This post-translational modification is crucial for regulating ciliary motility, particularly through its effects on specific inner-arm dyneins associated with the dynein regulatory complex (DRC) . Studies in Chlamydomonas have demonstrated that TTLL9-mediated polyglutamylation particularly affects the activity of dynein e, without significantly impacting other dynein types .

In which cellular compartments is TTLL9 typically found?

TTLL9 is present in multiple cellular compartments with varying concentrations:

• Cytoplasm: Contains approximately 10 times more TTLL9 than the axoneme

• Axoneme: Where TTLL9 performs its primary function of tubulin polyglutamylation

• Flagellar matrix: Present in lower amounts than in the axoneme

• Flagellar membrane fraction: Present in lower amounts than in the axoneme

The TTLL9-FAP234 complex formation is essential for the stability of TTLL9 in the cytoplasm, as demonstrated in studies where the absence of FAP234 led to the degradation of TTLL9 .

How conserved is TTLL9 across different organisms?

TTLL9 shows strong evolutionary conservation primarily among organisms possessing motile cilia or flagella. Below is a comparative table showing the distribution of TTLL9 and its binding partner FAP234 across various taxonomic groups:

This conservation pattern suggests that TTLL9 plays a specialized role in organisms with motile cilia, although some exceptions exist, such as C. elegans which possesses TTLL9 despite having only non-motile cilia .

What key characteristics should I look for when selecting a TTLL9 antibody for immunolocalization studies?

When selecting a TTLL9 antibody for immunolocalization in ciliary/flagellar studies, consider the following critical characteristics:

• Specificity: Select antibodies validated with appropriate controls, including TTLL9 knockout or knockdown samples. Research in Chlamydomonas and mice has utilized TTLL9-deficient mutants (tpg1 in Chlamydomonas and Ttll9-/- in mice) as negative controls .

• Cross-reactivity: Verify cross-reactivity with your species of interest. Given the conservation of TTLL9 across ciliated organisms, many antibodies may work across species, but validation is still necessary.

• Domain recognition: Choose antibodies recognizing conserved domains if working with multiple species, or species-specific regions for highly specific detection.

• Format compatibility: Ensure the antibody works in your intended application (immunofluorescence, immunoblotting, immunoprecipitation). Research shows TTLL9 can be successfully detected in various subcellular fractions using appropriate extraction methods .

• Detection sensitivity: Studies with Chlamydomonas demonstrated that hemagglutinin (HA)-tagged TTLL9 provides enhanced detection sensitivity, particularly for the low abundance of TTLL9 in flagellar membrane and matrix fractions .

How do I confirm the specificity of a TTLL9 antibody in my experimental system?

To validate TTLL9 antibody specificity, employ these methodological approaches:

• Genetic validation: The gold standard is testing the antibody on TTLL9 knockout or knockdown samples. Research has utilized Ttll9-/- mouse tissues or tpg1 mutant Chlamydomonas for this purpose .

• Western blot analysis: Perform western blotting on:

  • Wild-type samples (positive control)

  • TTLL9-deficient samples (negative control)

  • Recombinant TTLL9 protein (if available)

  Expected results should show a specific band at approximately 50 kDa for TTLL9 .

• Fractionation controls: Analyze different cellular fractions (cytoplasm, axoneme, flagellar matrix) to verify detection patterns consistent with known TTLL9 distribution. Research shows predominant localization in the cytoplasm and axoneme, with lower levels in flagellar matrix and membrane fractions .

• Co-immunoprecipitation: Verify the antibody's ability to co-precipitate known TTLL9 interactors, particularly FAP234, as demonstrated in Chlamydomonas studies using both anti-HA tag and anti-FAP234C antibodies .

• Immunofluorescence pattern analysis: Compare staining patterns with published localization data, focusing on axonemal structures in ciliated/flagellated cells.

What are the optimal extraction methods for detecting TTLL9 in different cellular compartments?

Different subcellular compartments require specific extraction protocols for optimal TTLL9 detection:

• Axonemal extraction:

  • Use high-salt extraction (0.6M NaCl) to solubilize TTLL9 from axonemes after demembranation with detergent

  • This method effectively releases TTLL9-FAP234 complex from the axoneme while preserving their association

  • Western blotting analysis of the extract should reveal both TTLL9 (~50 kDa) and FAP234 (~177 kDa) bands

• Flagellar matrix isolation:

  • Apply freeze-thaw methods rather than detergent extraction for more sensitive detection

  • This approach allows separation of matrix proteins from membrane and axonemal components

  • The freeze-thaw method has shown superior results for detecting the relatively small amounts of TTLL9 in the matrix fraction compared to traditional detergent-based methods

• Cytoplasmic extraction:

  • Use mild non-ionic detergents (e.g., NP-40) in physiological buffers

  • Include protease inhibitors to prevent degradation of TTLL9, as studies have indicated it may be unstable in the absence of FAP234

  • Consider adding phosphatase inhibitors if studying potential regulatory phosphorylation events

• Whole-cell extraction for comparative studies:

  • Direct lysis in SDS sample buffer provides good representation of total cellular TTLL9

  • When comparing TTLL9 levels across samples, standardize using housekeeping proteins for normalization

How can I properly design experiments to study the TTLL9-FAP234 complex formation?

To investigate TTLL9-FAP234 complex formation, implement these methodological approaches:

• Co-immunoprecipitation studies:

  • Use antibodies against either TTLL9 or FAP234 to pull down the complex

  • In research with Chlamydomonas, immunoprecipitation with anti-HA antibodies successfully co-precipitated FAP234 from samples expressing TTLL9-HA

  • Include appropriate controls: non-specific IgG, lysates from TTLL9 or FAP234 knockout models

• Sucrose density gradient analysis:

  • Perform sedimentation velocity measurements on high-salt extracts from axonemes

  • Both TTLL9 and FAP234 have been shown to co-migrate in fractions between 7S and 11S, confirming their presence in a single complex

  • Compare migration patterns with known molecular weight standards

  • Include samples from wild-type and mutant (tpg1, tpg2) organisms for comprehensive analysis

• Cross-linking experiments:

  • Use mild cross-linking agents to stabilize protein interactions prior to extraction

  • Optimize cross-linker concentration and reaction time to prevent non-specific aggregation

  • Analyze cross-linked products by SDS-PAGE followed by western blotting

• Recombinant protein interaction studies:

  • Express recombinant TTLL9 and FAP234 to study direct interactions in vitro

  • Use pull-down assays with tagged recombinant proteins to confirm direct binding

  • Consider truncation constructs to map interaction domains

• Yeast two-hybrid or mammalian two-hybrid assays:

  • These methods can provide additional confirmation of direct protein-protein interactions

  • Use full-length and domain-specific constructs to map interaction regions

How does TTLL9 knockout affect tubulin polyglutamylation patterns across different axonemal doublets?

TTLL9 knockout studies in mice and Chlamydomonas have revealed specific patterns of altered tubulin polyglutamylation across axonemal microtubule doublets:

What are the most sensitive methods for quantifying changes in polyglutamylation levels following TTLL9 manipulation?

For precise quantification of polyglutamylation changes in TTLL9 research, these methods offer optimal sensitivity:

• Immunoblotting with polyglutamylation-specific antibodies:

  • Use monoclonal antibodies specific for polyglutamate side chains (polyE)

  • Combine with α-tubulin antibodies (e.g., 12G10) to normalize for total tubulin content

  • Employ enhanced chemiluminescence (ECL) detection for greater sensitivity

  • Quantify signal intensity using densitometry software with appropriate background correction

• Mass spectrometry-based approaches:

  • Use targeted proteomic methods to quantify glutamylated peptides

  • MALDI-TOF or LC-MS/MS can detect specific mass shifts corresponding to different glutamate chain lengths

  • This approach provides both qualitative and quantitative data on the length of polyglutamate chains

  • Include internal standards for accurate quantification

• Immunofluorescence quantification:

  • Use confocal microscopy with consistent acquisition parameters

  • Perform line-scan analysis along axonemal structures for spatial distribution data

  • Apply deconvolution algorithms to improve spatial resolution

  • Use ratiometric imaging with total tubulin staining to normalize signal

• Immuno-electron microscopy with gold particle counting:

  • This approach allows doublet-specific quantification as used in studies of Ttll9-/- mice

  • Count gold particles per unit length of microtubule

  • Statistical analysis can identify significant changes in specific doublets

  • Combine with tomography for 3D spatial information

• Flow cytometry for population-level analysis:

  • Particularly useful for sperm or cell populations with flagella/cilia

  • Provides distribution data across large sample sizes

  • Can be combined with viability markers to correlate polyglutamylation with functional outcomes

How can I investigate the functional relationship between TTLL9-mediated polyglutamylation and specific dynein arm activity?

To study how TTLL9-mediated polyglutamylation affects specific dynein arm activities, implement these experimental approaches:

• Microtubule sliding assays:

  • Compare in vitro microtubule sliding velocities in axonemes from wild-type and TTLL9-deficient organisms

  • Research has shown that TTLL9 specifically affects dynein e activity, an inner-arm dynein associated with the dynein regulatory complex (DRC)

  • Use ATP-reactivated demembranated axonemes with varying ATP concentrations to generate force-velocity curves

• High-speed video microscopy of flagellar waveforms:

  • Record flagellar beating patterns using high-speed CCD cameras (>200 frames per second)

  • Analyze specific parameters: beat frequency, waveform amplitude, propagation velocity

  • Studies with Ttll9-/- mice revealed irregular beats with intervening stalls, suggesting specific effects on dynein coordination

  • Generate kymographs to visualize wave propagation defects

• Genetic interaction studies:

  • Create double mutants combining TTLL9 deficiency with mutations in specific dynein arms

  • For example, combine with outer dynein arm mutations (e.g., oda2 in Chlamydomonas)

  • Epistasis analysis can reveal functional relationships between polyglutamylation and specific dynein types

  • Previous studies combined oda2 and tpg1 mutations to investigate these interactions

• Cryo-electron tomography:

  • Compare 3D structures of wild-type and TTLL9-deficient axonemes

  • Focus on structural changes in inner dynein arms and the DRC

  • Correlate structural differences with functional defects

  • This approach can reveal how polyglutamylation affects dynein arm conformations

• Biochemical characterization of dynein ATPase activity:

  • Extract specific dynein types from wild-type and TTLL9-deficient axonemes

  • Measure ATPase activity under various conditions (ATP concentration, salt concentration)

  • Compare activity, processivity, and microtubule binding affinity

Why might I observe inconsistent TTLL9 antibody staining in immunofluorescence experiments?

Inconsistent TTLL9 staining in immunofluorescence may result from several methodological issues:

• Fixation-dependent epitope masking:

  • The TTLL9-FAP234 complex formation may be sensitive to fixation methods

  • Compare paraformaldehyde fixation with methanol or glutaraldehyde protocols

  • Include antigen retrieval steps (heat-mediated or enzymatic) if initial results are poor

  • Research indicates that TTLL9 exhibits different extraction properties in different compartments, suggesting fixation sensitivity

• Subcellular localization variability:

  • TTLL9 distributes differently between cytoplasm, axoneme, and flagellar matrix/membrane

  • The cytoplasmic pool is approximately 10 times larger than the axonemal pool

  • Ensure permeabilization protocols are optimized for accessing all cellular compartments

  • Use cellular fractionation controls to validate staining patterns

• Complex stability issues:

  • TTLL9 stability depends on FAP234 presence; in FAP234-deficient models, TTLL9 is rapidly degraded

  • Include protease inhibitors in all sample preparation steps

  • Consider using tagged TTLL9 constructs (e.g., TTLL9-HA) for enhanced detection, as demonstrated in Chlamydomonas studies

• Technical considerations:

  • Optimize antibody concentration through titration experiments

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal amplification methods such as tyramide signal amplification

  • Include positive controls (e.g., tissues known to express high levels of TTLL9)

  • Use TTLL9-deficient samples as negative controls

How can I address the challenge of detecting low-abundance TTLL9 in specific cellular compartments?

Detecting low-abundance TTLL9, particularly in flagellar matrix and membrane fractions, requires specialized approaches:

• Sample enrichment strategies:

  • Concentrate proteins from dilute samples using TCA precipitation or methanol/chloroform extraction

  • Pool multiple samples when working with flagellar fractions

  • Use tagged versions of TTLL9 (TTLL9-HA) for enhanced detection sensitivity, as demonstrated in Chlamydomonas research

• Enhanced detection methods for western blotting:

  • Employ highly sensitive chemiluminescent substrates (Super Signal West Femto)

  • Use cooled CCD camera-based imaging systems rather than film

  • Increase exposure times with anti-blooming technologies

  • Load higher protein amounts from low-abundance fractions

• Amplification-based immunofluorescence techniques:

  • Implement tyramide signal amplification (TSA) for significantly enhanced sensitivity

  • Use quantum dot-conjugated secondary antibodies for improved signal-to-noise ratios

  • Apply confocal microscopy with photomultiplier gain optimization

  • Consider structured illumination microscopy (SIM) for improved resolution

• Optimized subcellular fractionation protocols:

  • Use specialized extraction methods for different compartments:

    • High-salt extraction for axonemal TTLL9

    • Freeze-thaw methods for matrix fraction TTLL9

  • Research has shown that freeze-thaw methods provide superior sensitivity for detecting matrix-localized TTLL9 compared to traditional detergent-based approaches

• Immunoprecipitation followed by western blotting:

  • Concentrate TTLL9 from dilute samples via immunoprecipitation

  • Use high-affinity antibodies or anti-tag antibodies (anti-HA) for efficient capture

  • This approach has successfully detected TTLL9-FAP234 complexes in flagellar matrix fractions

How can TTLL9 antibodies be used to investigate ciliopathy mechanisms?

TTLL9 antibodies offer valuable research tools for investigating ciliopathy mechanisms through these methodological approaches:

• Comparative polyglutamylation analysis in ciliopathy models:

  • Compare tubulin polyglutamylation patterns between control and ciliopathy samples

  • Use TTLL9 antibodies alongside polyE antibodies to correlate enzyme levels with modification patterns

  • Research has shown that TTLL9 deficiency affects specific microtubule doublets differently, with doublet 5 showing the greatest reduction in polyglutamylation

• Fertility research applications:

  • TTLL9 knockout mice exhibit male infertility due to defective sperm motility

  • Analyze sperm samples from infertile males for TTLL9 expression and localization

  • Correlate TTLL9 levels with polyglutamylation patterns and specific flagellar ultrastructural features

  • Investigate potential TTLL9 mutations in cases of asthenozoospermia (reduced sperm motility)

• Primary ciliary dyskinesia (PCD) studies:

  • Examine TTLL9 expression and localization in respiratory epithelial cells from PCD patients

  • Correlate polyglutamylation patterns with specific ultrastructural defects in ciliary axonemes

  • The conservation of TTLL9 across ciliated organisms supports its potential role in human ciliary diseases

• Immunohistochemical analysis of tissue samples:

  • Apply TTLL9 antibodies to tissue microarrays containing ciliopathy and control samples

  • Focus on ciliated tissues: respiratory epithelium, ependymal cells, photoreceptors

  • Compare TTLL9 localization patterns with ciliary structural proteins

  • Correlate findings with clinical phenotypes and genetic data

• Rescue experiments in disease models:

  • Attempt phenotypic rescue through reintroduction of wild-type TTLL9 in cellular or animal models

  • Use antibodies to confirm successful expression and localization of the rescue construct

  • Monitor restoration of normal polyglutamylation patterns and ciliary function

What methodological considerations are important when analyzing TTLL9 expression in clinical samples?

When analyzing TTLL9 expression in clinical samples, implement these methodological approaches for optimal results:

• Sample preservation and processing:

  • Ciliary structures are fragile and can be lost during processing

  • Optimize fixation protocols to preserve both TTLL9 antigenicity and ciliary structures

  • For tissue biopsies, consider using both frozen and fixed samples in parallel analyses

  • When working with sperm samples, use swim-up techniques to isolate motile populations

• Controls and normalization strategies:

  • Include both positive controls (tissues with known TTLL9 expression) and negative controls

  • Use multiple reference genes/proteins for normalization in qPCR or western blot studies

  • Consider analyzing multiple TTLL family members (TTLL1-11) for comprehensive evaluation

  • Include analysis of FAP234 expression, as TTLL9 stability depends on this binding partner

• Correlation with functional assays:

  • Combine TTLL9 expression analysis with functional ciliary assessments:

    • High-speed video microscopy for ciliary beat frequency and waveform

    • Electron microscopy for ultrastructural analysis

  • Studies in Ttll9-/- mice demonstrated irregular flagellar beats with intervening stalls that correlate with infertility

• Genetic analysis integration:

  • Screen for TTLL9 mutations or polymorphisms in ciliopathy patients

  • Correlate protein expression patterns with genetic findings

  • Consider analyzing genes encoding TTLL9 interactors, particularly FAP234

  • TTLL9 and FAP234 are highly conserved in organisms with motile cilia, suggesting their importance in ciliary function

• Statistical considerations for clinical studies:

  • Calculate appropriate sample sizes based on expected effect sizes

  • Use paired analysis where possible (affected vs. unaffected tissues from same individual)

  • Account for potential confounding variables: age, sex, medication use, smoking status

  • Apply appropriate statistical tests for non-normally distributed data