Mol. Syst. Biol.

Extra chromosome copies markedly alter the physiology of eukaryotic cells, but the underlying reasons are not well understood. We created human trisomic and tetrasomic cell lines and determined the quantitative changes in their transcriptome and proteome in comparison with their diploid counterparts. We found that whereas transcription levels reflect the chromosome copy number changes, the abundance of some proteins, such as subunits of protein complexes and protein kinases, is reduced toward diploid levels. Furthermore, using the quantitative data we investigated the changes of cellular pathways in response to aneuploidy. This analysis revealed specific and uniform alterations in pathway regulation in cells with extra chromosomes. For example, the DNA and RNA metabolism pathways were downregulated, whereas several pathways such as energy metabolism, membrane metabolism and lysosomal pathways were upregulated. In particular, we found that the p62-dependent selective autophagy is activated in the human trisomic and tetrasomic cells. Our data present the first broad proteomic analysis of human cells with abnormal karyotypes and suggest a uniform cellular response to the presence of an extra chromosome

Phospho-regulation of the Shugoshin - Condensin interaction at the centromere in budding yeast.

Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin-condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast

A Conserved Role for SNX9-Family Members in the Regulation of Phagosome Maturation during Engulfment of Apoptotic Cells

Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution

Comparative analysis of the rhizosphere microbiota of Vaccinium corymbosum L. grown amateur and commercially in Lesser Poland Region

Borówka wysoka (Vaccinium corymbosum L.), znana jako borówka amerykańska, obecnie jest głównym gatunkiem borówki uprawianym w Europie i na świecie. Badania miały na celu analizę liczebności i składu gatunkowego mikroorganizmów zasiedlających strefę ryzosferową borówki uprawianej w różnych warunkach. Analizy ilościowe wykonano metodą seryjnych rozcieńczeń, a przynależność systematyczną drobnoustrojów oznaczono metodą Maldi Tof. Gleby, na których uprawiana była borówka, istotnie różniły się odczynem. Liczebność bakterii kształtowała się na poziomie 1·10³ do 5,7·10⁶, promieniowców od 1·10³ do 2,9·10⁶, grzybów 7·10⁴ do 7,2·10⁵ jtk·g⁻¹ s.m. gleby. Najlepszy wzrost wszystkich grup drobnoustrojów był obserwowany na podłożach o pH 6,5. Obecność Azotobacter sp. stwierdzono tylko w glebie o najwyższym pH (6.46). Zastosowane podłoża nie pozwoliły na wyizolowanie gatunków grzybów mykoryzowych typowych dla wrzosowatych. Wśród oznaczonych bakterii dominowały gatunki z rodzaju Bacillus, izolowano także przedstawicieli Pseudomonas, Pantonea, Lysinibacillus, Serratia, Citrobacter, Enterobacter, Solibacillus, Burkholderia i Azotobacter, a wśród promieniowców – Streptomyces. Grzyby reprezentowane były najliczniej przez rodzaje Trichoderma i Penicillium, stwierdzono również występowanie Aspergillus, Fusarium, Rhizopus, Microsporum, Phialophora i Rhodotorula.Highbush blueberry (Vaccinium corymbosum L.), known as the American blueberry, is now the main species of blueberries grown in Europe and the world. This plant has high requirements of soil and water, so during the summer months requires intensive irrigation and use of acidic substrate to the cultivation, because the normal development of plants can be carried out only in the presence of mycorrhizal fungi like Hymenoscyphus, Oidiodendron, Scytalidium, Myxotrichum, Clavaria and Gigaspora or Glomus. To accelerate the development and enhance of the plants is also worth to applied the vaccine containing selected strains of fungi forming mycorrhiza with plants, because their role is an enzymatic decomposition of organic matter, so that the plant receives easily digestible form of mineral nutrients. But very important is also the fact that a significant impact on the growth and yield of crops have all the microorganisms inhabiting the soil environment, especially those that live in the rhizosphere, and the population of saprophytic microbes may determine the success of the harvest and the size and quality of crops. The study aimed to analyze the abundance and species composition of microorganisms inhabiting the rhizosphere of blueberries grown in different conditions. Quantitative analysis was performed by serial dilution method on substrates of different pH value (4.5; 5.5 and 6.5), was determined the total count of bacteria, fungi, actinomycetes and indicator microorganisms of the genus Azotobacter. Taxonomy of microorganisms was determined by mass spectrometry method – Maldi Tof. Soils on which was cultivated blueberry differed significantly in site reaction. The number of bacteria was at a level of 1·10³ to 5.7·10⁶, actinomycetes from 1·10³ to 2.9·10⁶, fungi 7·10⁴ to 7.2·10⁵ cfu·g⁻¹ soil dry mass. The best growth of all microbial groups was observed in microbiological media having a pH of 6.5. The presence of Azotobacter sp. was found only in soil with a high pH (6.46). Among the identified bacteria dominated species of the genus Bacillus, also isolated were representatives of Pseudomonas, Pantonea, Lysinibacillus, Serratia, Citrobacter, Enterobacter, Solibacillus, Burkholderia and Azotobacter and among the actinomycetes Streptomyces. Fungi most frequently were represented by genus Trichoderma and Penicillium, the presence of Aspergillus, Fusarium, Rhizopus, Microsporum, Phialophora and Rhodotorula genus was also indicated. The species of mycorrhizal fungi, typical for ericaceous, were not isolated from tested soil samples

Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements.

Yeast vacuoles fragment and fuse in response to environmental conditions, such as changes in osmotic conditions or nutrient availability. Here we analyze osmotically induced vacuole fragmentation by time-lapse microscopy. Small fragmentation products originate directly from the large central vacuole. This happens by asymmetrical scission rather than by consecutive equal divisions. Fragmentation occurs in two distinct phases. Initially, vacuoles shrink and generate deep invaginations that leave behind tubular structures in their vicinity. Already this invagination requires the dynamin-like GTPase Vps1p and the vacuolar proton gradient. Invaginations are stabilized by phosphatidylinositol 3-phosphate (PI(3)P) produced by the phosphoinositide 3-kinase complex II. Subsequently, vesicles pinch off from the tips of the tubular structures in a polarized manner, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol-3,5-bisphosphate and the Fab1 complex. It is accelerated by the PI(3)P- and phosphatidylinositol 3,5-bisphosphate-binding protein Atg18p. Thus vacuoles fragment in two steps with distinct protein and lipid requirements

The CORVET Subunit Vps8 Cooperates with the Rab5 Homolog Vps21 to Induce Clustering of Late Endosomal Compartments

Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes