Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum.

The aim of this study was to investigate biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum. The sequence of a 10-kb region containing the capsule biosynthetic locus of Av. paragallinarum was determined. Two reference strains, i.e., 221 (serovar A) and H18 (serovar C), together with four Taiwanese field strains (all serovar C) were sequenced. The results showed that there are two genotypes (I and II) of the capsule biosynthetic locus in Av. paragallinarum, and the capsule genotype is independent of the serovar. The capsule biosynthetic loci of genotypes I and II consisted of six and five genes, respectively. The genotype I genes encoded proteins that are most similar to proteins from Pasteurella multocida capsule types A and F while the genotype II genes encoded proteins most similar to proteins from P. multocida capsule type D and Escherichia coli K5. The results suggested that genotype I strains contain hyaluronan or chondroitin in the capsule wall while genotype II contain heparosan. Enzymatic digestion of the capsule materials extracted from Av. paragallinarum showed that genotype I strains contained chondroitin while genotype II strains contained heparosan in the capsule. This is the first report on the existence of different genotypes of capsule biosynthesis genes in Av. paragallinarum and the presence of chondroitin and heparosan as chemical components of the capsule of Av. paragallinarum.

Actin-based motility during endocytosis in budding yeast.

Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility.

A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo.

BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family. RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex. CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.