A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Mechanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo.

Cilia regeneration is a physiological event, and while studied extensively in unicellular organisms, it remains poorly understood in vertebrates. In this study, using Xenopus multiciliated cells (MCCs) as a model, we demonstrate that, unlike unicellular organisms, deciliation removes the transition zone (TZ) along with the ciliary axoneme. While MCCs immediately begin the regeneration of the ciliary axoneme, surprisingly, the assembly of TZ was delayed. Instead, ciliary tip proteins, Sentan and Clamp, were the first to localize to regenerating cilia. Using cycloheximide (CHX) to block new protein synthesis, we show that the TZ protein B9d1 is not a component of the cilia precursor pool and requires new transcription/translation providing insights into the delayed repair of TZ. Moreover, CHX treatment led MCCs to assemble fewer (~ ten compared to ~150 in controls) but about wild-type length (78% of WT) cilia by gradually concentrating ciliogenesis proteins like IFT43 at a select few basal bodies, highlighting the exciting possibility of protein transport between basal bodies to facilitate faster regeneration in cells with multiple cilia. In summary, we demonstrate that MCCs begin regeneration with the assembly of ciliary tip and axoneme followed by TZ, questioning the importance of TZ in motile ciliogenesis.

Xenopus to the rescue: A model to validate and characterize candidate ciliopathy genes.

Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting millions of lives worldwide. Yet, our understanding of the gene regulatory networks that control cilia assembly and functions remain incomplete. With the advances in next-generation sequencing technologies, we can now rapidly predict pathogenic variants from hundreds of ciliopathy patients. While the pace of candidate gene discovery is exciting, most of these genes have never been previously implicated in cilia assembly or function. This makes assigning the disease causality difficult. This review discusses how Xenopus, a genetically tractable and high-throughput vertebrate model, has played a central role in identifying, validating, and characterizing candidate ciliopathy genes. The review is focused on multiciliated cells (MCCs) and diseases associated with MCC dysfunction. MCCs harbor multiple motile cilia on their apical surface to generate extracellular fluid flow inside the airway, the brain ventricles, and the oviduct. In Xenopus, these cells are external and present on the embryonic epidermal epithelia, facilitating candidate genes analysis in MCC development in vivo. The ability to introduce patient variants to study their effects on disease progression makes Xenopus a powerful model to improve our understanding of the underlying disease mechanisms and explain the patient phenotype.

Defects in the ratio of the dynein isoform, DHC11 in the long-flagella mutants of Chlamydomonas reinhardtii.

The long-flagella mutants (lf1, lf2, lf3 and lf4) of Chlamydomonas reinhardtii are defective in proteins that are required for the assembly of normal flagella, their phenotype being long flagella. In a previous study, we biophysically characterized these mutants for their waveform patterns, swimming speeds, beat frequencies and correlated these parameters with their flagellar lengths. We found an anomaly in this correlation and set out to explore the underlying molecular significance, if any. The diverse inner dynein isoforms are the flagellar motors that convert the chemical energy of ATP into the mechanical energy of motility; we probed the presence of one of these isoforms (DHC11, which might help in bend initiation) in the lf mutants and compared it with the wild-type. Our studies show that the ratio of DHC11 is defective in the long-flagella mutants of Chlamydomonas reinhardtii.

Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella.

BACKGROUND: Flagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical '9 + 2' cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain. RESULTS: We found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus. CONCLUSION: It appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved '9+2' axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26-57%) and beat frequencies (by 8-16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.