Organization and topology of photosystem I subunits

Intact spinach (Spinacia oleracea) thylakoid membranes were treated with various proteases and photosystem I (PSI) complexes were isolated from these membranes to define the membrane topology of specific PSI subunits. Trypsin treatment caused cleavage of the PSI-D and E subunits. Thermolysin treatment cleaved the PSI-D, E, H, and K subunits, and also caused limited degradation of the reaction center core PSI-A and B subunits. Pronase treatment produced the most dramatic results as the PSI-A and B subunits were cleaved to 47-, 45-, 26-, and 24-kilodalton products. In addition, pronase degraded the PSI-D, E, H, K, and L subunits. Proteolytic cleavage sites for several of the products were identified by amino acid sequencing. The results indicate that PSI-A, B, D, E, H, K, and L subunits all have stroma-exposed regions, and these findings are summarized in a model describing the subunit organization of PSI.     

The PCP effector Fuzzy controls cilial assembly and signaling by recruiting Rab8 and Dishevelled to the primary cilium

The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.     

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

Cutting edge: microbial products elicit formation of dendritic cell aggresome-like induced structures in macrophages

In response to a maturation stimulus, dendritic cells undergo the formation of ubiquitinated protein aggregates known as dendritic cell aggresome-like induced structures (DALIS). DALIS are thought to act as Ag storage structures, allowing for the prioritized degradation of proteins during infection. In this study, we demonstrate that murine macrophages can also form ubiquitinated protein aggregates that are indistinguishable from DALIS. These were formed in a dose- and time-dependent manner, and in response to a variety of microbial products. Surprisingly, the proteasome did not accumulate on these ubiquitinated protein structures, further underlining the difference between DALIS and aggresomes. Our studies suggest that DALIS formation is important for the function of Ag-presenting immune cells during infection.     

Cytogeography of the Phleum pratense group (Poaceae) in the Carpathians and Pannonia

Cytogeographical variability within the Phleum pratense group in the Carpathians and adjacent part of Pannonian lowland, based on 132 populations analysed by flow cytometry, is described. Only diploid and hexaploid plants were detected among 635 samples from the studied area. Diploids were found to be less frequent (127 plants, 20%) than hexaploids (508, 80%). With the exception of the single pure diploid population, diploids always co-occured with hexaploids (30 localities, 22.7%). The majority of populations (101, 76.5%) consisted of hexaploid plants. Most mixed populations occur in the Western Carpathians (26). In the Eastern Carpathians, mixed populations are much rarer, with three populations in Ukraine and one in Romania. In the Southern Carpathians, only hexaploids occur. The conventional taxonomic concept of the two species, diploid P. bertolonii and hexaploid P. pratense , was followed in spite of their sympatric occurence. Distribution maps based on chromosome number data from previous studies and on ploidy level estimates are given for both species in the studied area. The pattern of different distribution of the two taxa within the Carpathians is discussed.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 475–485.     

Identification of a viral antigen recognized by H-2-restricted cytolytic T lymphocytes on a murine leukemia virus-induced tumor

Monoclonal antibodies were produced against protein p30, a structural protein of murine leukemia viruses (MuLV) coded by the gag gene of MuLV. Three monoclonal antibodies of different isotypes (i.e., IgG-1, IgG-2a, and IgG-2b) were chosen for extensive analysis. These three antibodies bound to mouse tumor cells induced by Friend, Moloney, Rauscher, and Gross MuLV, but not to noninfected normal mouse spleen cells. The ability of these monoclonal antibodies to inhibit cytolytic T lymphocyte (CTL) activity by masking the antigens recognized by CTL on the target cell surface was studied in various CTL systems. It was found that the only CTL that were consistently inhibited in their lytic activity came from BALB.B (H-2b) mice immunized against syngeneic Gross MuLV-induced B.GV cells. These results thus showed that a subpopulation of BALB.B anti-Gross MuLV CTL recognized a Gross MuLV gag gene product expressed on the surface of B.GV cells.     

Incorporation of 32P and Growth of Pseudomonad UP-2 on n-Tetracosane

Cultures of the marine pseudomonad UP-2 growing on n-tetracosane contained both free cells and cells bound to the solid hydrocarbon. After separation by filtration through a Whatman no. 1 filter, the numbers of free and bound cells were estimated from the amount of ³²P incorporated into each fraction and the determined value of ³²P incorporation per viable cell in the filtrate (free cells). During the early exponential growth phase, over 80% of the cells were bound to large pieces of n-tetracosane; as the culture approached the stationary phase, the number of bound cells remained constant, whereas free cells continued to accumulate. Pulse-labeling experiments indicated that cells grew both on the surface of the solid and in the aqueous medium. During the growth cycle, a portion of the n-tetracosane which was initially nonfilterable was recovered in the filtrate in a form which was largely cell associated. This cell-associated n-tetracosane was preferentially utilized and could completely account for the observed growth of free cells.     

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.