<Emphasis Type="Italic">Chlamydomonas fla</Emphasis> mutants reveal a link between deflagellation and intraflagellar transport

Background

Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT.

Results

fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C), as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants.

Conclusions

Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active disassembly is distinct from the constitutive disassembly which plays a role in flagellar length control.

     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.     

The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist

Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.     

Complete genome sequence of Thermomonospora curvata type strain (B9)

Thermomonospora curvata Henssen 1957 is the type species of the genus Thermomonospora. This genus is of interest because members of this clade are sources of new antibiotics, enzymes, and products with pharmacological activity. In addition, members of this genus participate in the active degradation of cellulose. This is the first complete genome sequence of a member of the family Thermomonosporaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,639,016 bp long genome with its 4,985 protein-coding and 76 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Coraliomargarita akajimensis type strain (04OKA010-24)

Coraliomargarita akajimensis Yoon et al. 2007 is the type species of the genus Coraliomargarita. C. akajimensis is an obligately aerobic, Gram-negative, non-spore-forming, non-motile, spherical bacterium that was isolated from seawater surrounding the hard coral Galaxea fascicularis. C. akajimensis is of special interest because of its phylogenetic position in a genomically under-studied area of the bacterial diversity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Puniceicoccaceae. The 3,750,771 bp long genome with its 3,137 protein-coding and 55 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cell mitochondrial DNA

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin, and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands.Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.     

In vitro stimulation of rat liver cells by homologous partial hepatectomy serum

Summary A low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed. Supported by Grant 67-7 from the Illinois Division of the American Cancer Society.     

NUCLEAR-CYTOPLASMIC RELATIONS IN THE MITOSIS OF SEA URCHIN EGGS : III. γ-Ray-Induced Damage to Whole Eggs and Nucleate and Anucleate Half-Eggs

Sea urchin eggs were cut into halves. The nucleate and anucleate halves and whole eggs were irradiated with γ-rays and then fertilized with normal sperm. The first mitosis of the diploid half-egg was more delayed than the division of the whole egg. There was a small, but highly significant, delay of the mitosis of the haploid half-egg, thus demonstrating cytoplasmic sensitivity to ionizing radiation. Since the sensitivity of nucleate cells is influenced by cytoplasmic volume, the problem of the role of cytoplasm in repair is considered in relation to these data and other reports in the literature.