Effect of Nutrient Concentration and Salinity on Immotile–Motile Transformation of Chromera velia

ABSTRACT. Chromera velia (Chromerida: Alveolata) is a photosynthetic, unicellular organism closely related to parasitic apicomplexa. Diurnal rhythmicity of an immotile–motile transformation has been observed but its role in the life cycle remains largely unknown. Using a multiwell system, we show that salinity and f‐medium concentration significantly affect the percentage of motile C. velia cells. An inverse relationship between salinity and motility in C. velia occurred, and flagellation was also suppressed at high nutrient levels. These results suggest a low salinity environment with relatively low nutrient levels enables flagellate transformation during the diurnal cycle of C. velia.     

Distinct Mutants of Retrograde Intraflagellar Transport (IFT) Share Similar Morphological and Molecular Defects

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.     

ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.     

Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1–depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.     

Photosynthesis in Chromera velia Represents a Simple System with High Efficiency

Chromera velia (Alveolata) is a close relative to apicomplexan parasites with a functional photosynthetic plastid. Even though C. velia has a primitive complement of pigments (lacks chlorophyll c) and uses an ancient type II form of RuBISCO, we found that its photosynthesis is very efficient with the ability to acclimate to a wide range of irradiances. C. velia maintain similar maximal photosynthetic rates when grown under continual light-limited (low light) or light-saturated (high light) conditions. This flexible acclimation to continuous light is provided by an increase of the chlorophyll content and photosystem II connectivity under light limited conditions and by an increase in the content of protective carotenoids together with stimulation of effective non-photochemical quenching under high light. C. velia is able to significantly increase photosynthetic rates when grown under a light-dark cycle with sinusoidal changes in light intensity. Photosynthetic activities were nonlinearly related to light intensity, with maximum performance measured at mid-morning. C. velia efficiently acclimates to changing irradiance by stimulation of photorespiration and non-photochemical quenching, thus avoiding any measurable photoinhibition. We suggest that the very high CO₂ assimilation rates under sinusoidal light regime are allowed by activation of the oxygen consuming process (possibly chlororespiration) that maintains high efficiency of RuBISCO (type II). Despite the overall simplicity of the C. velia photosynthetic system, it operates with great efficiency.     

The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140^RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.     

The 3D Structure of the Apical Complex and Association with the Flagellar Apparatus Revealed by Serial TEM Tomography in Psammosa pacifica,a Distant Relative of the Apicomplexa

The apical complex is one of the defining features of apicomplexan parasites, including the malaria parasite Plasmodium, where it mediates host penetration and invasion. The apical complex is also known in a few related lineages, including several non-parasitic heterotrophs, where it mediates feeding behaviour. The origin of the apical complex is unclear, and one reason for this is that in apicomplexans it exists in only part of the life cycle, and never simultaneously with other major cytoskeletal structures like flagella and basal bodies. Here, we used conventional TEM and serial TEM tomography to reconstruct the three dimensional structure of the apical complex in Psammosa pacifica, a predatory relative of apicomplexans and dinoflagellates that retains the archetype apical complex and the flagellar apparatus simultaneously. The P. pacifica apical complex is associated with the gullet and consists of the pseudoconoid, micronemes, and electron dense vesicles. The pseudoconoid is a convex sheet consisting of eight short microtubules, plus a band made up of microtubules that originate from the flagellar apparatus. The flagellar apparatus consists of three microtubular roots. One of the microtubular roots attached to the posterior basal body is connected to bypassing microtubular strands, which are themselves connected to the extension of the pseudoconoid. These complex connections where the apical complex is an extension of the flagellar apparatus, reflect the ancestral state of both, dating back to the common ancestor of apicaomplexans and dinoflagellates.     

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly.     

Protein kinase involved in flagellar-length control

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.     

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

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.     

Flagellar length control system: testing a simple model based on intraflagellar transport and turnover

Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle.     

Ultrastructure and identification of the predatory flagellateColpodella pugnax Cienkowski (Apicomplexa) with a description ofColpodella turpis n. sp. and a review of the genus

A flagellated predator of the chlorophyte algaDunaliella spp. was examined by light and electron microscopy. Although this predator had previously been identified as a species of the kinetoplastid genusBodo, the present study revealed the flagellate to be indistinguishable fromColpodella pugnax, the type-species for its genus. The flagellate lacks a kinetoplast, a microtubule supported cytopharynx and paraxial rods in the flagella — characters universally found in kinetoplastid flagellates. The cell has mitochondria with vesicular cristae. Multiple membranes surround the cell and are underlain by longitudinal microtubules not originating from the flagellar region. Most notably, the flagellate has micropores and an apical complex including a conoid, sacculate rhoptries and, apparently, a polar ring. This study hs confirmed thatColpodella is the genus with free-living species most closely related to the apicomplexan parasites (i.e. the “Euapicomplexa” andPerkinsus). No unambiguous synapomorphy supports an “apicomplexan parasites” clade: Inclusion ofColpodella is necessary to secure the Apicomplexa as a monophyletic (=holophyletic) taxon. A new family, the Colpodellidae, is erected for this genus. Colpodella turpis, a previously undescribed species that also consumesDunaliella spp., was isolated from the same samples asC. pugnax. A diagnosis for this species is presented together with a brief review of the genus, in which we recognise seven species. The generic namesAlphamonas Aléxéieff,Nephromonas Droop andDingensia Patterson & Zölffel are rendered into synonomy withColpodella.     

A Highlights from MBoC Selection: A conserved flagella-associated protein in Chlamydomonas,FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.     

Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex

Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.     

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.     

High-resolution crystal structure and in vivo function of a kinesin-2 homologue in Giardia intestinalis

A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis-deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of G. intestinalis wild-type kinesin-2 (GiKIN2a) motor domain, with its docked neck linker and the hydrolysis-deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg(2+) at 1.6 and 1.8 A resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. G. intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes, with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in G. intestinalis.     

Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in Chlamydomonas flagella

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.     

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.     

Speed and Diffusion of Kinesin-2 Are Competing Limiting Factors in Flagellar Length-Control Model

Flagellar length control in Chlamydomonas is a tractable model system for studying the general question of organelle size regulation. We have previously proposed that the diffusive return of the kinesin motor that powers intraflagellar transport can play a key role in length regulation. Here, we explore how the motor speed and diffusion coefficient for the return of kinesin-2 affect flagellar growth kinetics. We find that the system can exist in two distinct regimes, one dominated by motor speed and one by diffusion coefficient. Depending on length, a flagellum can switch between these regimes. Our results indicate that mutations can affect the length in distinct ways. We discuss our theory’s implication for flagellar growth influenced by beating and provide possible explanations for the experimental observation that a beating flagellum is usually longer than its immotile mutant. These results demonstrate how our simple model can suggest explanations for mutant phenotypes.