Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.     

The DHC1b (DHC2) Isoform of Cytoplasmic Dynein Is Required for Flagellar Assembly

Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993–1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.     

Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.     

Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.     

多纤毛细胞纤毛精确组装的调控机制

高等动物体内气管、脑室管膜及输卵管等上皮组织具有一类富含运动纤毛的多纤毛细胞,通过其细胞表面运动纤毛的周期性摆动可以清洁气管、驱动脑脊液流动和受精卵运动.运动纤毛发生或功能的异常则可导致气管炎、脑积水、不孕不育等多种遗传疾病.然而,在多纤毛细胞分化过程中关于如何精确组装运动性纤毛复杂结构的分子机制仍不清楚.该研究运用蛋...     

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas

Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments (“F-actin”). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1–LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1–LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1–lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.     

Competition limits adaptation and productivity in a photosynthetic alga at elevated CO2

When competitive exclusion between lineages and genetic adaptation within lineages occur on the same timescale, the two processes have the potential to interact. I use experimental microbial evolution where strains of a photosynthetic microbe that differ in their physiological response to CO₂ enrichment are grown either alone or in communities for hundreds of generations under CO₂ enrichment. After about 300 generations of growth, strains that experienced competition while adapting to environmental change are both less productive and less fit than corresponding strains that adapted to that same environmental change in the absence of competitors. In addition, I find that excluding competitors not only limits that strain''s adaptive response to abiotic change, but also decreases community productivity; I quantify this effect using the Price equation. Finally, these data allow me to empirically test the common hypothesis that phytoplankton that are most able to take advantage of carbon enrichment in single-strain populations over the short term will increase in frequency within multi-strain communities over longer timescales.     

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38-40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.     

The Role of a Pollen-Expressed Cullin1 Protein in Gametophytic Self-Incompatibility in Solanum

We previously isolated a pollen factor, ui6.1, which encodes a Cullin1 protein (CUL1) that functions in unilateral interspecific incompatibility (UI) in Solanum. Here we show that CUL1 is also required for pollen function in self-incompatibility (SI). We used RNA interference (RNAi) to reduce CUL1 expression in pollen of Solanum arcanum, a wild SI tomato relative. Hemizygous T₀ plants showed little or no transmission of the transfer DNA (T-DNA) through pollen when crossed onto nontransgenic SI plants, indicating that CUL1-deficient pollen are selectively eliminated. When crossed onto a related self-compatible (SC) accession lacking active S-RNase, pollen transmission of the T-DNA followed Mendelian ratios. These results provide further evidence for functional overlap between SI and UI on the pollen side and suggest that CUL1 mutations will reinforce SI-to-SC transitions in natural populations only if preceded by loss of pistil S-RNase expression.     

Protein deficient chloroplast ribosomes in a yellow mutant of Chlamydomonas reinhardii

Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analyzed and compared by two-dimensional gel electrophoresis. The mixothrophically grown yellow-76 mutant differs from wild-type cells in lowered chlorophyll content and photosynthetic activity per chlorophyll unit. The latter is connected with the decreased activity of the ribulose-I,5-diphosphate-carboxylase enzyme. Analytical ultracentrifugation of cell extracts shows a normal amount of free 70S ribosomes and 50S subunit in the mutant cells. Two-dimensional gel electrophoresis shows considerable alterations in the protein composition of the 70S ribosomes of the mutant. Two proteins are absent from the electrophoretograms of the yellow-76 mutant, and seven proteins are present in reduced amounts. The genetical analysis shows a Mendelian pattern of inheritance, indicating that protein alterations presumably are localized in nuclear DNA.Abbreviation MNNG N-methyl-N-nitro-N-nitrosoguanidine     

The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.     

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.     

The Cytosolic DnaJ-like Protein Djp1p Is Involved Specifically in Peroxisomal Protein Import

The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ. DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes. Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome. The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities. Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal. Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant. We conclude that Djp1p is specifically required for peroxisomal protein import.