The mammalian retromer protein complex, which consists of three proteins -Vps26, Vps29, and Vps35 -in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

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Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence of B. pseudomallei 08 to cultured cells increases dramatically following prior growth at 30°C or less compared to that following prior growth at 37°C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27°C but not at 37°C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene, pilA, is essential for microcolony development by B. pseudomallei 08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although the B. pseudomallei genome sequence strain, K96243, also contains transcriptionally active pilA, microcolony formation rarely occurs following growth at either 27°C or 37°C and cell association occurs significantly less than with strain 08. Analysis of pilA transcription in 08 identified that pilA is dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription of pilA in K96243 differed from that in 08. Our study also suggests that biofilm formation by B. pseudomallei 08 and K96243 on polyvinylchloride is not mediated by pilA. Adherence and microcolony formation, and pilA transcription, vary between strains, consistent with known genomic variation in B. pseudomallei, and these phenotypes may be relevant to colonization from the environment.

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo-(binds KAP3A, B56␣, and ASEF) and ␤-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of ␤-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of ␤-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of ␤-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with ␤-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only ␤-catenin stimulated APC anchorage at the membrane. Our findings identify ␤-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

We have established an in vitro model of long-term continuous Chlamydia pneumoniae infection in HEp-2 cells. Using transmission electron microscopy, we demonstrated the presence of spontaneous abnormal chlamydial inclusions similar in appearance to the persistent chlamydial forms induced in vitro by treatment with cytokines or antibiotics or by nutrient deprivation.Chlamydia pneumoniae is a frequent cause of communityacquired pneumonia and bronchitis in adults and children. Like other chlamydial species, it can cause prolonged or chronic infections which may persist for months or years (11). These persistent infections have been implicated in the development of a number of chronic diseases, including chronic obstructive pulmonary disease, atherosclerosis, and asthma (20). However, whether persistent C. pneumoniae is a cause, a triggering cofactor, or an innocent bystander remains controversial.Persistent chlamydial infections can be established in vitro using several methods, including treatment with cytokines (2, 4, 16, 18) or antibiotics (5, 6) or by deprivation of certain nutrients (13). In all cases they have been described as having morphologically abnormal reticulate bodies (RBs), which suggests that they are somehow altered during their otherwise normal development.In the present study we describe the ultrastructural findings determined using an in vitro model of long-term continuous C. pneumoniae infection in HEp-2 cells, a respiratory epithelial cell line (14,17).Briefly, confluent HEp-2 cells were inoculated once with C. pneumoniae isolate TW-183 (ATCC VR2282) or CM-1 (ATCC VR1360) to achieve 100% infection. After 3 to 5 days, when lysis of most of the infected host cells was seen, the culture medium was replaced with fresh medium but without added cycloheximide. After 1 week of further incubation, growth of colonies of new host cells was observed. Since then, continuous C. pneumoniae cultures have been maintained for over 4 years by reseeding the infected host cells into new flasks to prevent overgrowth. No new cells or chlamydiae were added. C. pneumoniae remained viable and cultivable in this model.Two days prior to sampling, the continuously infected cells were seeded onto six-well plates. Cell monolayers were trypsinized, and infected cells were collected into 1.5-ml centrifuge tubes and fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. Samples were prepared for transmission electron microscopy by standard procedures (9). Samples were postfixed in osmium tetroxide, followed by uranyl acetate. The cells were then dehydrated in increasing concentrations of ethanol (50, 70, and 90%) and acetone (90 and 100%) and subsequently embedded in Spurr's epoxy resin. Ultrathin sections (50 to 100 nm in thickness) were prepared and collected onto 200-mesh copper grids, contrasted with 1% uranyl acetate and Reynolds lead citrate before being examined, and photographed using a JEOL 1200EX transmission electron microscope.Three types of chlamydial inclusions were observed by transmission el...

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56␣, -␤, and -⑀ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56␣ subcellular localization. We detected B56␣ in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56␣. Conversely, CRM1 overexpression shifted B56␣ to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451-469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56␣. Active NESs were identified at similar positions in the cytoplasmic B56-␤ and ⑀ isoforms, but not in the nuclear-localized B56-␦ or ␥ isoforms. The transient expression of B56␣ induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56␣ co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56␣-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56␣ can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.Reversible protein phosphorylation is a key mechanism regulating a myriad of cellular processes. A delicate balance between the opposing effects of protein kinases and phosphatases determines the functional state of many proteins. Protein phosphatase 2A (PP2A) 3 refers to a major family of heterotrimeric serine-threonine phosphatase enzymes in the cell (1-3). The core enzyme is a dimer consisting of a 36-kDa catalytic C-subunit and a 65-kDa structural regulatory A-subunit, which acts as a scaffold to bring into proximity the C-subunit and protein substrates bound by the diverse regulatory B-subunits. There are four B-subunit gene families each with multiple genes that encode a range of splice variant peptides. The diverse nature of PP2A is inherent in its composition, which is potentially comprised of over 200 distinct protein complexes each containing different combinations of the A-, B-, and C-subunits, and hence allowing for variability and subtle regulation in phosphatase action (3).The B-subunits are postulated to regulate PP2A activity in different ways: (a) by targeting the holoenzyme to specific subcellular locations (e.g. B55␣ directs PP2A to microtubules (4)), (b) determining substrate specificity (e.g. PP2A complexes containing B55 or B72 B-subunits cause the activation or inhibition of SV40 DNA replication, respectively, because of differences in substrate recognition sites on...

SummaryBurkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1g), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensisinfected cells. The expression by B. pseudomalleiinfected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis.Analysis of dentine resorption by B. pseudomalleiinduced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.

We have investigated the adherence of Burkholderia pseudomallei, cultured under a number of different conditions, to six human epithelial cell lines. While several complex medium compositions had relatively little effect on adherence, growth at 30°C was found to significantly increase adherence to all cell lines relative to that of cultures grown at 37°C (P < 0.001).