Specific α- and β-Tubulin Isotypes Optimize the Functions of Sensory Cilia in Caenorhabditis elegans

Primary cilia have essential roles in transducing signals in eukaryotes. At their core is the ciliary axoneme, a microtubule-based structure that defines cilium morphology and provides a substrate for intraflagellar transport. However, the extent to which axonemal microtubules are specialized for sensory cilium function is unknown. In the nematode Caenorhabditis elegans, primary cilia are present at the dendritic ends of most sensory neurons, where they provide a specialized environment for the transduction of particular stimuli. Here, we find that three tubulin isotypes—the α-tubulins TBA-6 and TBA-9 and the β-tubulin TBB-4—are specifically expressed in overlapping sets of C. elegans sensory neurons and localize to the sensory cilia of these cells. Although cilia still form in mutants lacking tba-6, tba-9, and tbb-4, ciliary function is often compromised: these mutants exhibit a variety of sensory deficits as well as the mislocalization of signaling components. In at least one case, that of the CEM cephalic sensory neurons, cilium architecture is disrupted in mutants lacking specific ciliary tubulins. While there is likely to be some functional redundancy among C. elegans tubulin genes, our results indicate that specific tubulins optimize the functional properties of C. elegans sensory cilia.THE fitness of all organisms depends on an ability to appropriately sense and respond to the environment. At the cellular level, many specific architectures have evolved to optimize these sensory functions. Prominent among these is the sensory cilium, a tubulin-based cytoplasmic extension that interrogates the extracellular environment in many biological contexts (Davenport and Yoder 2005; Berbari et al. 2009). Cilia are important for the transduction of a broad range of visual, auditory, mechanical, thermal, and chemical stimuli. They also function during development to receive a variety of signals, both chemical and mechanical, that regulate proliferation and differentiation (Goetz and Anderson 2010). Indeed, the disruption of cilium assembly and function can give rise to a spectrum of human diseases collectively known as ciliopathies (Berbari et al. 2009; Lancaster and Gleeson 2009). These disorders, which include autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), Bardet–Biedl syndrome, Meckel–Gruber syndrome, and Joubert syndrome, are associated with a variety of pathogenic conditions including polycystic kidneys and neurological impairments.At the core of all cilia and flagella is the microtubule axoneme. This characteristic structural element comprises nine doublet outer microtubules that may surround a central pair, the presence of which often indicates a motile cilium/flagellum. Like all microtubule-based structures, ciliary axonemes are built of heterodimers of α- and β-tubulins, highly conserved small GTP-binding proteins. The recruitment of other cilium components, including signal transduction machinery, requires a conserved assembly and maintenance process called intraflagellar transport (IFT) (Blacque et al. 2008; Pedersen and Rosenbaum 2008). IFT employs two major complexes that transport ciliary cargo bidirectionally by traveling along the axonemal microtubules. Loss of individual IFT components can cause a broad spectrum of defects in the assembly, maintenance, and function of cilia.Important insights into cilium structure and function have come from studies of genetically tractable organisms, particularly the green alga Chlamydomonas and the nematode Caenorhabditis elegans (Bae and Barr 2008; Pedersen and Rosenbaum 2008). In C. elegans, sensory cilia are found exclusively at the dendritic ends of sensory neurons. These cilia constitute a highly specialized sensory environment characterized by localized sensory receptors and specific signaling components. Cilium morphology is quite distinctive in many of these cells and likely contributes to their functional specialization (Ward et al. 1975). Recent progress has shed light on the mechanisms that confer this specialization onto more general pan-ciliary pathways (Evans et al. 2006; Mukhopadhyay et al. 2007; Jauregui et al. 2008; Mukhopadhyay et al. 2008; Silverman and Leroux 2009).The genomes of many eukaryotes harbor multiple α- and β-tubulin genes. Two hypotheses, which are not mutually exclusive, have been proposed to account for these paralogs (Cleveland 1987; Wade 2007). At one extreme, different tubulin isotypes might be functionally redundant, such that their minor coding differences are largely irrelevant. According to this model, multiple genes allow the maintenance of a stable pool of available monomers and dimers. The small amount of sequence variation within the α- and β-tubulin families supports this idea, as do studies of functionally redundant mitotic tubulins in C. elegans (Ellis et al. 2004; Lu et al. 2004; Phillips et al. 2004; Lu and Mains 2005). The alternative hypothesis proposes that specific structures, e.g., ciliary axonemes or axonal microtubules, rely on tubulins optimized for specific roles. Support for this idea has come from studies of cultured mammalian neurons (Joshi and Cleveland 1989), Drosophila (Hutchens et al. 1997; Raff et al. 1997), and human tubulins (Vent et al. 2005; Jaglin et al. 2009). In Drosophila, studies of motile sperm flagella have revealed that the sperm-specific β2 tubulin isoform builds not only the specialized motile axoneme but also all other tubulin-based structures (Kemphues et al. 1982). However, sequences both within and outside the axoneme motif in the C-terminal tail of this tubulin isoform are required for the flagellar axoneme, and other closely related β-tubulins cannot support this role (Fuller et al. 1987; Raff et al. 1997; Popodi et al. 2008). Genetic interactions have provided evidence that β2 tubulin heterodimerizes with the α-tubulin 84B (Hays et al. 1989), which also possesses specific functional properties not provided by structurally similar α-tubulins (Hutchens et al. 1997). In C. elegans, a specific role for tubulin isoforms has been described in the six touch receptor neurons. These nonciliated cells harbor unusual 15-filament microtubules composed of dimers of the α-tubulin MEC-12 and the β-tubulin MEC-7. The loss of mec-7 or mec-12, the expression of which is largely restricted to these cells, results in the conversion of 15-filament microtubules to the standard 11-microfilament variety and a commensurate loss of light-touch response (Savage et al. 1989; Fukushige et al. 1999; Bounoutas et al. 2009). Thus experimental support exists for both of these opposing views, and it seems likely that the role of specific tubulin isoforms in regulating microtubule structure and function differs according to cell and organelle type.The C. elegans genome encodes nine α- and six β-tubulin genes (Gogonea et al. 1999). Some of these genes, particularly tba-1, tba-2, tbb-1, and tbb-2, are expressed broadly during embryogenesis and function redundantly in spindle assembly and positioning (Ellis et al. 2004; Lu et al. 2004; Phillips et al. 2004; Lu and Mains 2005). tba-1 and tbb-2 have also been recently shown to be important for axon outgrowth and synaptogenesis (Baran et al. 2010). Several others, including mec-7, mec-12, and the β-tubulin ben-1, have been identified through genetic screens for particular phenotypes, such as touch insensitivity or benzimidazole resistance (Driscoll et al. 1989; Savage et al. 1989; Fukushige et al. 1999). However, the extent to which specific tubulin isoforms are required for structural and functional diversity in the C. elegans nervous system remains unknown. Here, taking advantage of several existing genome-wide data sets, we identify the α-tubulins TBA-6 and TBA-9 and the β-tubulin TBB-4 as strong candidates for tubulins that have roles in sensory cilia. We find that each of these genes are expressed in characteristic, partially overlapping, sets of sensory neurons, where their products localize to ciliary axonemes. While the loss of any one (or all three) of these genes does not abolish ciliogenesis, tubulin mutants exhibit significant defects in the localization of cilium proteins and in some cilium-dependent behavioral responses. Together, our results indicate that specific α- and β-tubulin isoforms are important, although not essential, for the efficient assembly and function of specific classes of C. elegans sensory cilia. Sensory cilia throughout the animal kingdom may therefore employ specific tubulin isoforms to optimize their function.     

H-NS mediates the silencing of laterally acquired genes in bacteria

Histone-like nucleoid structuring protein (H-NS) is a modular protein that is associated with the bacterial nucleoid. We used chromatin immunoprecipitation to determine the binding sites of H-NS and RNA polymerase on the Salmonella enterica serovar Typhimurium chromosome. We found that H-NS does not bind to actively transcribed genes and does not co-localize with RNA polymerase. This shows that H-NS principally silences gene expression by restricting the access of RNA polymerase to the DNA. H-NS had previously been shown to preferentially bind to curved DNA in vitro. In fact, at the genomic level we discovered that the level of H-NS binding correlates better with the AT-content of DNA. This is likely to have evolutionary consequences because we show that H-NS binds to many Salmonella genes acquired by lateral gene transfer, and functions as a gene silencer. The removal of H-NS from the cell causes un-controlled expression of several Salmonella pathogenicity islands, and we demonstrate that this has deleterious consequences for bacterial fitness. Our discovery of this novel role for H-NS may have implications for the acquisition of foreign genes by enteric bacteria.     

In vitro methods for culturing vertebrate and mosquito stages of Plasmodium

The development of in vitro culture systems for the vertebrate stages of Plasmodium led to major advancements in malaria research. Here we review both improvements made in these techniques and the recent achievement of the in vitro growth of mosquito stages from ookinete to infective sporozoite.     

Seaweed biogeochemistry: Global assessment of C:N and C:P ratios and implications for ocean afforestation

Algal carbon-to-nitrogen (C:N) and carbon-to-phosphorus (C:P) ratios are fundamental for understanding many oceanic biogeochemical processes, such as nutrient flux and climate regulation. We synthesized literature data (444 species, >400 locations) and collected original samples from Tasmania, Australia (51 species, 10 locations) to update the global ratios of seaweed carbon-to-nitrogen (C:N) and carbon-to-phosphorus (C:P). The updated global mean molar ratio for seaweed C:N is 20 (ranging from 6 to 123) and for C:P is 801 (ranging from 76 to 4102). The C:N and C:P ratios were significantly influenced by seawater inorganic nutrient concentrations and seasonality. Additionally, C:N ratios varied by phyla. Brown seaweeds (Ochrophyta, Phaeophyceae) had the highest mean C:N of 27.5 (range: 7.6–122.5), followed by green seaweeds (Chlorophyta) of 17.8 (6.2–54.3) and red seaweeds (Rhodophyta) of 14.8 (5.6–77.6). We used the updated C:N and C:P values to compare seaweed tissue stoichiometry with the most recently reported values for plankton community stoichiometry. Our results show that seaweeds have on average 2.8 and 4.0 times higher C:N and C:P than phytoplankton, indicating seaweeds can assimilate more carbon in their biomass for a given amount of nutrient resource. The stoichiometric comparison presented herein is central to the discourse on ocean afforestation (the deliberate replacement of phytoplankton with seaweeds to enhance the ocean biological carbon sink) by contributing to the understanding of the impact of nutrient reallocation from phytoplankton to seaweeds under large-scale seaweed cultivation.     

PRODUCTION OF HYALINE HAIRS BY INTERTIDAL SPECIES OF FUCUS (FUCALES) AND THEIR ROLE IN PHOSPHATE UPTAKE1

A field study to determine the precise times of year at which three intertidal species of Fucus start to produce hyaline hairs and cease producing such hairs was conducted on the Isle of Man, U.K. Hairs were first observed during February, and within 6 days of their initial appearance, all tagged plants of all species at all tidal heights on the shore possessed hairs. Hair production continued until the beginning of October, at which time Fucus plants growing at the lowest stations (+ 3.0 m) had glabrous apical growth. Hair production continued later into the year for plants growing higher on the shore, and it was not until mid-November that glabrous apical growth was observed in all plants. Phosphate uptake rates of pilose (hairy) and glabrous (hairless) apical sections were measured in November 1988 for F. spiralis L. and in January 1989 for F. spiralis and F. serratus L., at phosphate concentrations ranging from 0.8 μM (ambient seawater) to 9.0 μM. In ambient seawater, pilose plants of F. spiralis removed phosphate 2–3 times faster than glabrous plants, whereas the uptake rates of pilose plants of F. serratus were about 50% greater than those of glabrous plants. The differences between uptake rates of pilose and glabrous plants of both species were smaller or nonsignificant at higher phosphate concentrations. The field and laboratory data are consistent with the hypothesis that hairs are formed in Fucus as a response to increased nutrient demand and that hairs facilitate the uptake of nutrients from seawater at concentrations typical of natural situations.     

Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance.

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].