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.

Recently Published

  • Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control

    Patel, Mansi B; Griffin, Paul J; Olson, Spencer F; Dai, Jin; Hou, Yuqing; Malik, Tara; Das, Poulomi; Zhang, Gui; Zhao, Winston; Witman, George B.; et al. (2024-03-08)
    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

    Hou, Yuqing; Cheng, Xi; Witman, George B (2022-12-09)
    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.
  • Consensus nomenclature for dyneins and associated assembly factors

    Braschi, Bryony; Omran, Heymut; Witman, George B.; Pazour, Gregory J.; Pfister, K. Kevin; Bruford, Elspeth A.; King, Stephen M. (2022-01-10)
    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

    Cai, Kai; Zhao, Yanhe; Zhao, Lei; Phan, Nhan; Hou, Yuqing; Cheng, Xi; Witman, George B.; Nicastro, Daniela (2021-11-01)
    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

    Hou, Yuqing; Zhao, Lei; Kubo, Tomohiro; Cheng, Xi; McNeill, Nathan A.; Oda, Toshiyuki; Witman, George B. (2021-06-28)
    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

    Craft Van De Weghe, Julie; Harris, J. Aaron; Kubo, Tomohiro; Witman, George B.; Lechtreck, Karl F. (2020-09-11)
    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

    Picariello, Tyler; Hou, Yuqing; Kubo, Tomohiro; McNeill, Nathan A.; Yanagisawa, Haru-Aki; Oda, Toshiyuki; Witman, George B. (2020-05-13)
    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

    Zhao, Lei; Hou, Yuqing; McNeill, Nathan A.; Witman, George B. (2019-12-30)
    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

    Fu, Gang; Zhao, Lei; Dymek, Erin; Hou, Yuqing; Song, Kangkang; Phan, Nhan; Shang, Zhiguo; Smith, Elizabeth F.; Witman, George B.; Nicastro, Daniela (2019-10-31)
    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

    Zhao, Lei; Hou, Yuqing; Picariello, Tyler; Craige, Branch; Witman, George B. (2019-06-03)
    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.
  • A global analysis of IFT-A function reveals specialization for transport of membrane-associated proteins into cilia

    Picariello, Tyler; Brown, Jason M.; Hou, Yuqing; Swank, Gregory; Cochran, Deborah A.; King, Oliver D.; Lechtreck, Karl; Pazour, Gregory J.; Witman, George B. (2019-02-11)
    Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140. This article has an associated First Person interview with the first author of the paper.
  • A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

    Kubo, Tomohiro; Hou, Yuqing; Cochran, Deborah A.; Witman, George B.; Oda, Toshiyuki (2018-05-01)
    Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here, we show using cryo-electron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the fbeta head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.
  • The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

    Hou, Yuqing; Witman, George B. (2017-09-01)
    Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multi-subunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here, we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B, but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that IFT46, ODA16, and the outer dynein arm interact for IFT of the latter.
  • IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium

    Brown, Jason M.; Picariello, Tyler; Cochran, Deborah A.; Witman, George B.; Lechtreck, Karl (2017-05-31)
    Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.
  • Characterization of a new oda3 allele, oda3-6, defective in assembly of the outer dynein arm-docking complex in Chlamydomonas reinhardtii

    Brown, Jason M.; Mosley, Matthew; Montes-Berrueta, Daniela; Hou, Yuqing; Yang, Fan; Scarbrough, Chasity; Witman, George B.; Wirschell, Maureen (2017-03-14)
    We have used an insertional mutagenesis approach to generate new C. reinhardtii motility mutants. Of 56 mutants isolated, one is a new allele at the ODA3 locus, called oda3-6. Similar to the previously characterized oda3 alleles, oda3-6 has a slow-jerky swimming phenotype and reduced swimming speed. The oda3-6 mutant fails to assemble the outer dynein arm motor and outer dynein arm-docking complex (ODA-DC) in the ciliary axoneme due to an insertion in the 5' end of the DCC1 gene, which encodes the DC1 subunit of the ODA-DC. Transformation of oda3-6 with the wild-type DCC1 gene rescues the mutant swimming phenotype and restores assembly of the ODA-DC and the outer dynein arm in the cilium. This is the first oda3 mutant to be characterized at the molecular level and is likely to be very useful for further analysis of DC1 function.
  • Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

    Kubo, Tomohiro; Brown, Jason; Bellve, Karl D.; Craige, Branch; Craft, Julie M.; Fogarty, Kevin E.; Lechtreck, Karl-Ferdinand; Witman, George B. (2016-05-15)
    The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.
  • Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

    San Agustin, Jovenal T.; Pazour, Gregory J.; Witman, George B. (2015-12-01)
    Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88(-/-) mice are completely sterile. They produce approximately 350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.
  • CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice

    McKenzie, Casey W.; Craige, Branch; Kroeger, Tiffany V.; Finn, Rozzy; Wyatt, Todd A.; Sisson, Joseph H.; Pavlik, Jacqueline A.; Strittmatter, Lara; Hendricks, Gregory M.; Witman, George B.; et al. (2015-09-15)
    Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.
  • Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone

    Yang, T. Tony; Su, Jimmy; Wang, Won-Jing; Craige, Branch; Witman, George B.; Tsou, Meng-Fu Bryan.; Liao, Jung-Chi (2015-09-14)
    The transition zone (TZ) of primary cilia serves as a diffusion barrier to regulate ciliogenesis and receptor localization for key signaling events such as sonic hedgehog signaling. Its gating mechanism is poorly understood due to the tiny volume accommodating a large number of ciliopathy-associated molecules. Here we performed stimulated emission depletion (STED) imaging of collective samples and recreated superresolved relative localizations of eight representative species of ciliary proteins using position averages and overlapped with representative electron microscopy (EM) images, defining an architectural foundation at the ciliary base. Upon this framework, transmembrane proteins TMEM67 and TCTN2 were accumulated at the same axial level as MKS1 and RPGRIP1L, suggesting that their regulation roles for tissue-specific ciliogenesis occur at a specific level of the TZ. CEP290 is surprisingly localized at a different axial level bridging the basal body (BB) and other TZ proteins. Upon this molecular architecture, two reservoirs of intraflagellar transport (IFT) particles, correlating with phases of ciliary growth, are present: one colocalized with the transition fibers (TFs) while the other situated beyond the distal edge of the TZ. Together, our results reveal an unprecedented structural framework of the TZ, facilitating our understanding in molecular screening and assembly at the ciliary base.
  • DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B. (2015-08-01)
    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo-electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC-dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

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