A new periplasmic protein of Escherichia coli which is synthesized in spheroplasts but not in intact cells.

The gene spy from Escherichia coli has been cloned and sequenced. It encodes a precursor of a so far unknown 139-residue, rather basic periplasmic protein. It was not detectable immunologically in intact cells but was produced abundantly in spheroplasts. It could be a stress protein specific for spheroplasting.     

Chemical composition of the attachment pad secretion of the locust Locusta migratoria

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C₁₆ and C₂₀ in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.     

The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis in Arabidopsis thaliana

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.     

<Emphasis Type="Italic">Mycosphaerella podagrariae</Emphasis>—a necrotrophic phytopathogen forming a special cellular interaction with its host <Emphasis Type="Italic">Aegopodium podagraria</Emphasis>

We present a new kind of cellular interaction found between Mycosphaerella podagrariae and Aegopodium podagraria, which is remarkably different to the interaction type of the obligate biotrophic fungus Cymadothea trifolii, another member of the Mycosphaerellaceae (Capnodiales, Dothideomycetes, Ascomycota) which we have described earlier. Observations are based on both conventional and cryofixed material and show that some features of this particular interaction are better discernable after chemical fixation. We were also able to generate sequences for nuclear ribosomal DNA (complete SSU, 5.8 S and flanking ITS-regions, D1–D3 region of the LSU) confirming the position of M. podagrariae within Mycosphaerellaceae.     

Plant cytokinesis requires de novo secretory trafficking but not endocytosis

During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.     

Rootletin forms centriole-associated filaments and functions in centrosome cohesion

After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.     

Human cytomegalovirus entry into dendritic cells occurs via a macropinocytosis-like pathway in a pH-independent and cholesterol-dependent manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection.     

Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration

BACKGROUND INFORMATION: Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. RESULTS AND CONCLUSIONS: Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.     

The <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response regulator ARR22 is a putative AHP phospho-histidine phosphatase expressed in the chalaza of developing seeds

Background  

The Arabidopsis response regulator 22 (ARR22) is one of two members of a recently defined novel group of two-component system (TCS) elements. TCSs are stimulus perception and response modules of prokaryotic origin, which signal by a His-to-Asp phosphorelay mechanism. In plants, TCS regulators are involved in hormone response pathways, such as those for cytokinin and ethylene. While the functions of the other TCS elements in Arabidopsis, such as histidine kinases (AHKs), histidine-containing phosphotransfer proteins (AHPs) and A-type and B-type ARRs are becoming evident, the role of ARR22 is poorly understood.