Brucella "hitches a ride" with autophagy

Autophagy involves lysosomal-mediated degradation of cellular components and contributes to host immunity. Some pathogens avoid autophagy-mediated killing, while others exploit it to acquire host cell nutrients. Starr et?al. reveal that the intracellular bacterial pathogen Brucella abortus can "hitch a ride" with autophagy, subverting autophagy machinery to spread from cell to cell (Starr et?al., 2012).     

Plant programmed cell death: can't live with it; can't live without it

The decision of whether a cell should live or die is fundamental for the wellbeing of all organisms. Despite intense investigation into cell growth and proliferation, only recently has the essential and equally important idea that cells control/programme their own demise for proper maintenance of cellular homeostasis gained recognition. Furthermore, even though research into programmed cell death (PCD) has been an extremely active area of research there are significant gaps in our understanding of the process in plants. In this review, we discuss PCD during plant development and pathogenesis, and compare/contrast this with mammalian apoptosis.     

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein (Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high Mr polypeptides of 20 S dynein. This antiserum reacted in "Western" immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.     

Structural comparison of purified dynein proteins with in situ dynein arms

Using the quick-freeze deep-etch technique, we describe the structure of outer-arm dynein proteins from Chlamydomonas and Tetrahymena after adsorption to a mica surface, after high-salt dissociation, and after glutaraldehyde fixation, and compare these images to the configuration of outer arms bound to microtubules. After adsorption to mica, the extracted dyneins from both organisms look like three-headed “bouquets”, as reported for Tetrahymena by Johnson & Wall (1983b). High magnification images demonstrate that each head carries a slender “stalk” and a long “stem”, and that small subunits decorate the stems and create a “flowerpot” domain at the base of the bouquet. Exposure to high salt induces this trimer to dissociate into a two-headed species and a single-headed species; it also stimulates the decorative elements to dissociate from the stems. Dynein is thus constructed on the same general plan as myosin, with large globular heads, narrow stems and additional small subunits that associate with the stems. The splayed-out image of the bouquet appears to be a distortion arising during adsorption to mica since, after brief glutaraldehyde fixation, the three heads remain closely associated as vertices of a triangular unit. In situ, the three heads also adopt this trigonal configuration. Two of the three are visible from the exterior of the axoneme and constitute the bilobed rigor head we described previously (Goodenough & Heuser, 1982). The third head faces the interior of the axoneme where, we propose, it forms the “hook” of the outer arm as seen in thin section. We further propose that the decorative elements associated with the stem coalesce to form the two outer-arm “feet” seen in situ, and that at least one of the in vitro stalks is equivalent to the in situ stalk, which extends from the head to the B microtubule. Deep-etch images of stretched axonemes, partially extracted axonemes, and dynein-decorated brain microtubules indicate that each outer arm, as traditionally viewed, is a hybrid of two dynein molecules: its two feet derive from one molecule, whereas its trigonal head derives from the molecule located distally. The resultant overlapping configuration creates the diagonal “linkers” seen in situ, which correspond to the in vitro stems. Thus, a row of dynein arms is essentially a dynein polymer that extends from the tip to the base of a doublet microtubule, each head riding on its neighbor's feet like a row of circus elephants. Such dynein-dynein interactions may account for the co-operativity of dynein-microtubule binding, and may play an important role in generating ciliary motility.     

Carrier of Wingless (Cow), a Secreted Heparan Sulfate Proteoglycan,Promotes Extracellular Transport of Wingless

Morphogens are signaling molecules that regulate growth and patterning during development by forming a gradient and activating different target genes at different concentrations. The extracellular distribution of morphogens is tightly regulated, with the Drosophila morphogen Wingless (Wg) relying on Dally-like (Dlp) and transcytosis for its distribution. However, in the absence of Dlp or endocytic activity, Wg can still move across cells along the apical (Ap) surface. We identified a novel secreted heparan sulfate proteoglycan (HSPG) that binds to Wg and promotes its extracellular distribution by increasing Wg mobility, which was thus named Carrier of Wg (Cow). Cow promotes the Ap transport of Wg, independent of Dlp and endocytosis, and this function addresses a previous gap in the understanding of Wg movement. This is the first example of a diffusible HSPG acting as a carrier to promote the extracellular movement of a morphogen.     

Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function

In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.     

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.     

Water hyacinth in Lake Victoria: Why did it vanish so quickly and will it return?

Water hyacinth has been a cause of great concern in terms of environmental and socio-economic impacts within Lake Victoria. In the late 1990s however it rapidly disappeared but since the causes are unknown its possible return cannot be predicted. Growth chamber and laboratory experiments investigating CO₂ assimilation and growth rate found that different phenotypic, density-acclimated, growth forms of water hyacinth behaved differently. PI-curves and changes in biomass revealed that short bulbous (SB) growth forms took up CO₂ more rapidly and increased in biomass quicker than tall non-bulbous (TN) growth forms. This allows the two growth forms to flourish within two different niches. One, the SB form, as a colonising opportunist and the other as a taller plant that attempts to avoid self-shading in dense mats. Light is an important limiting factor to water hyacinth growth. Light becomes non-limiting to CO₂ uptake at a PAR of ≈2000 μE m⁻² s⁻¹. In Lake Victoria this light level occurs for about 6 h around midday. Plant growth is thus light limited for most of the day and can be limited even at midday during cloudy weather. Although weevils likely played a role in the rapid disappearance of water hyacinth, its demise was too rapid and synchronous in this large lake for weevils to be solely responsible. The cloudy, wet El Nino weather of 1997/1998 was probably a major contributory factor to poor growth that led to the reduction in water hyacinth biomass lake-wide. Currently within Lake Victoria an improved light climate, an ever increasing supply of nutrients and a potentially unstable weevil population will likely allow the resurgence of this aggressive weed.     

Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.     

Analysis of mammalian dynein using antibodies against a polypeptides of sea urchin sperm flagellar dynein

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.     

Hitching a ride to the top: peroxisomes fuel cilium with cholesterol

正Primary cilium, which protrudes from the cell, is a microtubule-based structure ensheathed by a highly specialized plasma membrane (PM). It serves as a signaling hub for sensing and transducing various external stimuli including fluids, light, odorants as well as extracellular molecules.