Witman Lab

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https://hdl.handle.net/20.500.14038/203

This collection showcases the journal articles and other publications authored by researchers in the Witman Lab at UMass Chan Medical School. The Witman Lab joined the Department of Radiology in 2017 from the former Department of Cell and Developmental Biology.

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Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control
(2024-03-08) Patel, Mansi B; Griffin, Paul J; Olson, Spencer F; Dai, Jin; Hou, Yuqing; Malik, Tara; Das, Poulomi; Zhang, Gui; Zhao, Winston; Witman, George B.; Lechtreck, Karl F; Radiology; Witman Lab
Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP. KAP dissociates from IFT at the ciliary tip and diffuses back to the cell body. This observation led to the diffusion-as-a-ruler model of ciliary length control, which postulates that KAP is progressively sequestered into elongating cilia because its return to the cell body will require increasingly more time, limiting motor availability at the ciliary base, train assembly, building block supply, and ciliary growth. Here, we show that Chlamydomonas FLA8 also returns to the cell body by diffusion. However, more than 95% of KAP and FLA8 are present in the cell body and, at a given time, just ~1% of the motor participates in IFT. After repeated photobleaching of both cilia, IFT of fluorescent kinesin subunits continued indicating that kinesin-2 cycles from the large cell-body pool through the cilia and back. Furthermore, growing and full-length cilia contained similar amounts of kinesin-2 subunits and the size of the motor pool at the base changed only slightly with ciliary length. These observations are incompatible with the diffusion-as-a-ruler model, but rather support an "on-demand model," in which the cargo load of the trains is regulated to assemble cilia of the desired length.
Direct in situ protein tagging in Chlamydomonas reinhardtii utilizing TIM, a method for CRISPR/Cas9-based targeted insertional mutagenesis
(2022-12-09) Hou, Yuqing; Cheng, Xi; Witman, George B.; Radiology; Witman Lab
Chlamydomonas reinhardtii is an important model organism for the study of many cellular processes, and protein tagging is an increasingly indispensable tool for these studies. To circumvent the disadvantages of conventional approaches in creating a tagged cell line, which involve transforming either a wild-type or null-mutant cell line with an exogenous DNA construct that inserts randomly into the genome, we developed a strategy to tag the endogenous gene in situ. The strategy utilizes TIM, a CRISPR/Cas9-based method for targeted insertional mutagenesis in C. reinhardtii. We have tested the strategy on two genes: LF5/CDKL5, lack of which causes a long-flagella phenotype, and Cre09.g416350/NAP1L1, which has not been studied previously in C. reinhardtii. We successfully tagged the C-terminus of wild-type LF5 with the hemagglutinin (HA) tag with an efficiency of 7.4%. Sequencing confirmed that these strains are correctly edited. Western blotting confirmed the expression of HA-tagged LF5, and immunofluorescence microscopy showed that LF5-HA is localized normally. These strains have normal length flagella and appear wild type. We successfully tagged the N-terminus of Cre09.g416350 with mNeonGreen-3xFLAG with an efficiency of 9%. Sequencing showed that the tag region in these strains is as expected. Western blotting confirmed the expression of tagged protein of the expected size in these strains, which appeared to have normal cell size, growth rate, and swimming speed. This is the first time that C. reinhardtii endogenous genes have been edited in situ to express a wild-type tagged protein. This effective, efficient, and convenient TIM-tagging strategy promises to be a useful tool for the study of nuclear genes, including essential genes, in C. reinhardtii.
Dynein and Intraflagellar Transport
(2012-01-01) Witman, George B.; Department of Cell and Developmental Biology
This chapter provides a brief background on intraflagellar transport (IFT) and reviews the studies culminating in the identification of the dynein motor that powers retrograde IFT. IFT is the active movement of multi-subunit particles along axonemal doublet microtubules in the space between the outer-doublet microtubules and the membrane of cilia and flagella. Following this, it describes the known subunits of this dynein, discussing their specific functions, and examining how they fit together in the intact motor. Furthermore, it discusses the function of the dynein in recycling IFT proteins and other flagellar components, in transporting signals from the cilium to the cell body, in ciliary maintenance, and so on. Finally, it states that the basic function of cytoplasmic dynein 2 as the motor for retrograde IFT is now well established, and its general molecular architecture can be accurately predicted. However, much remains to be learned about this unique and important dynein. The currently intense focus on investigating ciliary function and assembly (and disassembly) is likely to provide more information on the role of dynein 2 in the transport of specific proteins and signals out of the cilium. Whether dynein 2 functions as a microtubule motor in other places where IFT particles are found besides the cilium is an important question that has yet to be addressed.
The Chlamydomonas Flagellum as a Model for Human Ciliary Disease
(2009-01-01) Pazour, Gregory J.; Witman, George B.; Department of Cell Biology; Program in Molecular Medicine
Studies in Chlamydomonas have illuminated the basic biology of many ciliary and basal body proteins involved in human diseases and developmental disorders, including PCD, hydrocephalus, epilepsy, situs inversus, and PKD. This chapter reviews past work on Chlamydomonas that has provided important insights into these diseases, and discusses additional diseases for which Chlamydomonas has the potential to be very informative in the future. Frequently, the human genes causing a disease have been identified by positional cloning, and the proteins they encode have been shown to be localized to cilia or basal bodies by immunofluorescence microscopy, but the functions of the proteins are understood poorly or not at all.ChlamydomonasIn many of these cases, Chlamydomonas offers the best opportunity for a detailed analysis that could lead to an understanding of the basic biology of the human disease protein. Diseases associated with them usually involve defects in the ciliary assembly machinery or ciliary signaling. These diseases include polycystic kidney disease (PKD), retinal degeneration and blindness, and several syndromes that usually involve kidney disease and/or blindness plus other symptoms. These diseases are explained in detail. Genomic stability is dependent on correct centriole duplication and function and control of the cell cycle may involve the primary cilium, so defects in cilia and basal bodies/centrioles also may lead to cancer, which is also covered here.
A unified taxonomy for ciliary dyneins
(2011-10-01) Hom, Erik F. Y.; Witman, George B.; Harris, Elizabeth H.; Dutcher, Susan K.; Kamiya, Ritsu; Mitchell, David R.; Pazour, Gregory J.; Porter, Mary E.; Sale, Winfield S.; Wirschell, Maureen; Yagi, Toshiki; King, Stephen M.; Program in Molecular Medicine; Department of Cell Biology
The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.
High-speed digital imaging of ependymal cilia in the murine brain
(2009-01-01) Lechtreck, Karl-Ferdinand; Sanderson, Michael J.; Witman, George B.; Department of Physiology; Department of Cell Biology
The development and health of mammals requires proper ciliary motility. Ciliated epithelia are found in the airways, the uterus and Fallopian tubes, the efferent ducts of the testes, and the ventricular system of the brain. A technique is described for the motion analysis of ependymal cilia in the murine brain. Vibratome sections of the brain are imaged by differential interference contrast microscopy and recorded by high-speed digital imaging. Side views of individual cilia are traced to establish their bending pattern. Tracking of individual cilia recorded in top view allows determination of bend planarity and beat direction. Ciliary beat frequency is determined from line scans of image sequences. The capacity of the epithelium to move fluid and objects is revealed by analyzing the velocity of polystyrene beads added to brain sections. The technique is useful for detailed assessment of how various conditions or mutations affect the fidelity of ciliary motility at the ependyma. The methods are also applicable to other ciliated epithelia, for example, in airways.
HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B
(2009-08-22) Lechtreck, Karl-Ferdinand; Luro, Scott; Awata, Junya; Witman, George B.; Department of Cell Biology
Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella
(2009-01-01) Engel, Benjamin D.; Lechtreck, Karl-Ferdinand; Sakai, Tsuyoshi; Ikebe, Mitsuo; Witman, George B.; Marshall, Wallace F.; Department of Physiology; Department of Cell Biology
The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies.
IC97 is a novel intermediate chain of I1 dynein that interacts with tubulin and regulates interdoublet sliding
(2009-07-08) Wirschell, Maureen; Yang, Chun; Yang, Pinfen; Fox, Laura; Yanagisawa, Haru-aki; Kamiya, Ritsu; Witman, George B.; Porter, Mary E.; Sale, Winfield S.; Department of Cell Biology
Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein--a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both alpha- and beta-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.
Isolation of Chlamydomonas flagella
(2013-06-01) Craige, Branch; Brown, Jason M.; Witman, George B.; Department of Cell and Developmental Biology
A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub-fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol.
CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content
(2010-09-08) Craige, Branch; Tsao, Che-Chia; Diener, Dennis R.; Hou, Yuqing; Lechtreck, Karl-Ferdinand; Rosenbaum, Joel L.; Witman, George B.; Department of Cell Biology
Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.
The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella
(2009-12-30) Lechtreck, Karl-Ferdinand; Johnson, Eric C.; Sakai, Tsuyoshi; Cochran, Deborah A.; Ballif, Bryan A.; Rush, John; Pazour, Gregory J.; Ikebe, Mitsuo; Witman, George B.; Program in Molecular Medicine; Department of Physiology; Department of Cell Biology
In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.
Consensus nomenclature for dyneins and associated assembly factors
(2022-01-10) Braschi, Bryony; Omran, Heymut; Witman, George B.; Pazour, Gregory J.; Pfister, K. Kevin; Bruford, Elspeth A.; King, Stephen M.; Witman Lab; Program in Molecular Medicine; Division of Cell Biology and Imaging, Department of Radiology
Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.
Structural organization of the C1b projection within the ciliary central apparatus
(2021-11-01) Cai, Kai; Zhao, Yanhe; Zhao, Lei; Phan, Nhan; Hou, Yuqing; Cheng, Xi; Witman, George B.; Nicastro, Daniela; Division of Cell Biology and Imaging, Department of Radiology
Motile cilia have a '9+2' structure containing nine doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic, proteomic and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas reinhardtii with those of three CA mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA, where they interact with the candidate CA protein FAP413. FAP42 is a large protein that forms the peripheral 'beam' of the C1b projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b projections, and loss of these proteins leads to ciliary motility defects.
Chlamydomonas FAP70 is a component of the previously uncharacterized ciliary central apparatus projection C2a
(2021-06-28) Hou, Yuqing; Zhao, Lei; Kubo, Tomohiro; Cheng, Xi; McNeill, Nathan A.; Oda, Toshiyuki; Witman, George B.; Witman Lab; Division of Cell Biology and Imaging, Department of Radiology
Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and approximately 36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70 (CFAP70 in humans), which also has been reported to be associated with the doublet microtubules. Here, we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the C2a projection of the CA. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 (CFAP65 in humans) and FAP147 (MYCBPAP in humans). Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65 and FAP147 as the first defining components of the C2a projection.
Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas
(2020-09-11) Craft Van De Weghe, Julie; Harris, J. Aaron; Kubo, Tomohiro; Witman, George B.; Lechtreck, Karl F.; Witman Lab; Department of Radiology
Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP-beta-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-beta-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by approximately 90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-beta-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP-beta-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.
TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii
(2020-05-13) Picariello, Tyler; Hou, Yuqing; Kubo, Tomohiro; McNeill, Nathan A.; Yanagisawa, Haru-Aki; Oda, Toshiyuki; Witman, George B.; Witman Lab; Division of Cell Biology and Imaging, Department of Radiology
Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.
The unity and diversity of the ciliary central apparatus
(2019-12-30) Zhao, Lei; Hou, Yuqing; McNeill, Nathan A.; Witman, George B.; Witman Lab; Division of Cell Biology and Imaging, Department of Radiology
Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The central pair of microtubules plus associated projections, termed the central apparatus (CA), is involved in the control of flagellar motility and is essential for the normal movement of '9+2' cilia. Research using the green alga Chlamydomonas reinhardtii, an important model system for studying cilia, has provided most of our knowledge of the protein composition of the CA, and recent work using this organism has expanded the number of known and candidate CA proteins nearly threefold. Here we take advantage of this enhanced proteome to examine the genomes of a wide range of eukaryotic organisms, representing all of the major phylogenetic groups, to identify predicted orthologues of the C. reinhardtii CA proteins and explore how widely the proteins are conserved and whether there are patterns to this conservation. We also discuss in detail two contrasting groups of CA proteins-the ASH-domain proteins, which are broadly conserved, and the PAS proteins, which are restricted primarily to the volvocalean algae. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus
(2019-10-31) Fu, Gang; Zhao, Lei; Dymek, Erin; Hou, Yuqing; Song, Kangkang; Phan, Nhan; Shang, Zhiguo; Smith, Elizabeth F.; Witman, George B.; Nicastro, Daniela; Department of Radiology, Division of Cell Biology and Imaging
Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large ( > 2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.
Proteome of the central apparatus of a ciliary axoneme
(2019-06-03) Zhao, Lei; Hou, Yuqing; Picariello, Tyler; Craige, Branch; Witman, George B.; Witman Lab; Division of Cell Biology and Imaging, Department of Radiology
Nearly all motile cilia have a "9+2" axoneme containing a central apparatus (CA), consisting of two central microtubules with projections, that is essential for motility. To date, only 22 proteins are known to be CA components. To identify new candidate CA proteins, we used mass spectrometry to compare axonemes of wild-type Chlamydomonas and a CA-less mutant. We identified 44 novel candidate CA proteins, of which 13 are conserved in humans. Five of the latter were studied more closely, and all five localized to the CA; therefore, most of the other candidates are likely to also be CA components. Our results reveal that the CA is far more compositionally complex than previously recognized and provide a greatly expanded knowledge base for studies to understand the architecture of the CA and how it functions. The discovery of the new conserved CA proteins will facilitate genetic screening to identify patients with a form of primary ciliary dyskinesia that has been difficult to diagnose.

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