Cilia locally synthesize proteins to sustain their ultrastructure and functions

Cilia are microtubule-based hair-like organelles propelling locomotion and extracellular liquid flow or sensing environmental stimuli. As cilia are diffusion barrier-gated subcellular compartments, their protein components are thought to come from the cell body through intraflagellar transport or diffusion. Here we show that cilia locally synthesize proteins to maintain their structure and functions. Multicilia of mouse ependymal cells are abundant in ribosomal proteins, translation initiation factors, and RNA, including 18 S rRNA and tubulin mRNA. The cilia actively generate nascent peptides, including those of tubulin. mRNA-binding protein Fmrp localizes in ciliary central lumen and appears to function in mRNA delivery into the cilia. Its depletion by RNAi impairs ciliary local translation and induces multicilia degeneration. Expression of exogenous Fmrp, but not an isoform tethered to mitochondria, rescues the degeneration defects. Therefore, local translation defects in cilia might contribute to the pathology of ciliopathies and other diseases such as Fragile X syndrome.

Toxicity and Tissue Accumulation Characteristics of the Herbicide Pendimethalin in Ginger (Zingiber Officinale Roscoe)

BackgroundEnvironmental health and food safety issues potentially caused by the dinitroaniline herbicide pendimethalin are a worldwide concern. Due to its importance for crop plants, the determination of possible toxicity and accumulation characteristics of pendimethalin in ginger should be determined. ResultsThe toxicity response of ginger and tissue accumulation effects of pendimethalin on ginger biomass were studied by utilizing pendimethalin in a dose-response study. No significant effect on ginger biomass is observed when the concentration of pendimethalin used is less than 6.7 ppm, while > 10 ppm pendimethalin significantly reduces the biomass of ginger. This is attributed to root damage. The net photosynthetic rate of ginger when treated with 16.7 ppm pendimethalin is 11.37% lower than that of the control organisms, which is mainly caused by stomatal limitation. In addition, high-dose pendimethalin (16.7 ppm) causes the accumulation of reactive oxygen species (ROS) in ginger. The activity of superoxide dismutase (SOD) and peroxidase increases accordingly, maintaining the dynamic balance of ROS content. There is no significant effect on malondialdehyde levels or on membrane permeability. Pendimethalin has no significant effect on the expression of ginger α-tubulin mRNA. The damage of high-dose (16.7 ppm) pendimethalin to ginger is mainly caused by oxidative stress. Pendimethalin is significantly accumulated in ginger roots, but not rhizomes. ConclusionsBecause of this, application of pendimethalin to treat weeds in ginger fields may not pose a threat to human health.

Effect of ischemic postconditioning on cell apoptosis and expression of relevant genes in non-culprit coronary arteries

This study was performed to determine the effect of ischemic postconditioning on cell apoptosis and angiotensin II receptor type 1 (AT1), connexin 43 (Cx43), and β-tubulin mRNA expression in non-culprit arteries. Non-culprit arterial tissues were isolated from a rabbit myocardial ischemia-reperfusion model and randomly divided into sham, ischemia-reperfusion, and ischemic postconditioning groups. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Expression of angiotensin II, AT1, Cx43, and β-tubulin mRNA was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). TUNEL analysis indicated significantly higher ratios of apoptotic cells in the ischemia-reperfusion group than in the sham group. However, significantly fewer apoptotic cells were observed in the ischemic postconditioning group than in the ischemia-reperfusion group. The qRT-PCR results indicated significantly higher expression of AT1, Cx43, and β-tubulin mRNA in the ischemia-reperfusion group than in the sham group. However, expression of AT1, Cx43, and β-tubulin was lower in the ischemic postconditioning group than in the ischemia-reperfusion group. The ratios of apoptotic cells and mRNA expression of AT1, Cx43, and β-tubulin in non-culprit arteries were increased after ischemia-reperfusion. Ischemic postconditioning may decrease these features and inhibit the progression of non-culprit arteries.

Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues

The localization, mass, and dynamics of microtubules are important in many processes. Cells may actively monitor the state of their microtubules and respond to perturbation, but how this occurs outside mitosis is poorly understood. We used gene expression analysis in quiescent cells to analyze responses to subtle and strong perturbation of microtubules. Genes encoding α-, β, and γ-tubulins, but not δ- or ε-tubulins, exhibited the strongest differential expression response to microtubule-stabilizing versus destabilizing drugs. Q-PCR of exon versus intron sequences confirmed that these changes were caused by regulation of tubulin mRNA stability and not transcription. Using tubulin mRNA stability as a signature to query the GEO database, we find that tubulin genes respond to toxins known to damage microtubules. Importantly, we find many other experimental perturbations, including multiple signaling and metabolic inputs that trigger tubulin differential expression, suggesting their novel role in the regulation of microtubule cytoskeleton. Mechanistic follow up confirms that one important physiological signal, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, indeed regulates tubulin mRNA stability via changes in microtubule dynamics. We propose that that tubulin gene expression is regulated as part of many coordinated biological responses, with wide implications in physiology and toxicology. Furthermore, we present a new way to discover microtubule regulation using genomics.

Stress-Induced Hsp70 Gene Expression and Inactivation of Cryptosporidium parvum Oocysts by Chlorine-Based Oxidants

ABSTRACT Our research on the mechanisms of action of chlorine-based oxidants on Cryptosporidium parvum oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on C. parvum oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of β-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.

Cloning and sequencing of tubulin cDNAs from Artemia franciscana: evidence for differential expression of α- and β-tubulin genes

Tubulin is a heterodimeric protein composed of α- and β-tubulin. In most organisms, they are encoded by multiple gene families whose members are subject to differential regulation. The objective of the work described herein was to better understand tubulin gene expression in the extremophile Artemia franciscana To this end tubulin cDNAs were cloned and sequenced. αAT2, an α-tubulin cDNA, differed by one nucleotide from αAT1, a previously cloned Artemia cDNA. This change, possibly generated by allelic variation, caused an M313V substitution in α-tubulin. The amino acid sequence of β-tubulin encoded by βAT1, one of only a very limited number of cloned crustacean β-tubulin cDNA sequences yet available, and the first from Artemia, was similar to other β-tubulins. However, βAT1 possessed four degenerate TATA boxes in the 5′ untranslated region, although authentic TATA and CCAAT boxes occurred in the 3′ non-coding sequence. Analyses by quantitative PCR demonstrated that the amount of tubulin mRNA declined relative to total mRNA in progressive life history stages of Artemia and also that the organism contained more αAT2- than βAT1-tubulin mRNA at all developmental phases examined.

Global Regulation of the Interphase Microtubule System by Abundantly Expressed Op18/Stathmin

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer–polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer–polymer partitioning, enables Op18 to exert global regulation of the microtubule system.