REMODELING OF THE SECRETORY PATHWAY AND CELL POLARITY MACHINERY IN DIVIDING AND ENVIRONMENTALLY STRESSED SCHIZOSACCHAROMYCES POMBE CELLS

Ph.DDOCTOR OF PHILOSOPH

Nucleocytoplasmic Shuttling of the TACC Protein Mia1p/Alp7p Is Required for Remodeling of Microtubule Arrays during the Cell Cycle

Microtubule arrays are remodeled as cells proceed through the cell cycle. It is important to understand how remodeling is regulated in time and space. In fission yeast, the conserved microtubule associated TACC/TOG complex plays an important role in organizing microtubules throughout the cell cycle. Here we show that this complex undergoes nucleocytoplasmic shuttling through the nuclear import and export signals located in the TACC protein Mia1p/Alp7p. When the Crm1p-dependent nuclear export signal of Mia1p is disabled, Mia1p accumulates in the nucleus while its partner protein Alp14p/TOG is restricted to the cytoplasm. This leads to defects in assembly of both interphase arrays and the mitotic spindle. Artificial targeting of Alp14p to the nucleus partially rescues the mitotic spindle defects caused by lack of Mia1p nuclear export. Interestingly, the nuclear export sequence of Mia1p appears to overlap with the Alp14p binding site. We propose that intricate regulation of the subcellular distribution of TACC/TOG complexes drives microtubule array remodeling as cells progress through the cell cycle

Model for analysis of environmental impacts of production processes in flooring industry based on LCA

Proizvodnja podnih obloga je, s aspekta utjecaja na okoliš, vrlo kompleksan proces zbog velikog broja utjecajnih parametara - različitih kemijskih emisija, neminovnosti otpada, većeg broja utjecajnih fizičkih veličina poput buke i vibracija, prašine i zračenja, zatim potrošnje energije, recikljivosti itd. Zanimanje za primjenu LCA u području proizvodnje podnih obloga traje već skoro dva desetljeća i postaje sve intenzivnije. Tijekom tog razdoblja evidentirani su različiti pristupi i pokušaji realizacije LCA u ovom širokom području. U ovom radu predstavljen je opći model za ocjenjivanje utjecaja na okoliš procesa proizvodnje podnih obloga zasnovan na LCA. Razvijeni model predstavlja doprinos unapređenju stanja u području i dodatni korak ka standardizaciji LCA analiza u području proizvodnje podnih obloga. Verifikacija je realizirana kroz studiju slučaja utemeljenog na realnom industrijskom postrojenju za proizvodnju PVC podnih obloga.The production of floor coverings, in terms of environmental impact, is a very complex process due to a large number of influential parameters – various chemical emissions, inevitability of waste, a great number of influential physical quantities such as noise and vibration, dust and radiation, energy consumption, recyclability, etc. Interest in the application of LCA within the production of floor coverings has been present for almost two decades and is intensely increasing. During this period different approaches and attempts of LCA implementation have been recorded in this broad field. This paper presents a general model for environmental impact assessment of the production process of floor coverings based on LCA. The developed model represents a contribution to the improvement and a further step towards the standardization of LCA analysis in this field of floor covering production. Verification has been carried out through a case study based on an industrial facility which produces PVC floor coverings

Community review: a robust and scalable selection system for resource allocation within open science and innovation communities

Resource allocation is essential to the selection and implementation of innovative projects in science and technology. With large stakes involved in concentrating large fundings over a few promising projects, current “winner-take-all” models for grant applications are time-intensive endeavours that mobilise significant researcher time in writing extensive project proposals, and rely on the availability of a few time-saturated volunteer experts. Such processes usually carry over several months, resulting in high effective costs compared to expected benefits. Faced with the need for a rapid response to the COVID-19 pandemic in 2020, we devised an agile “community review” system, similar to distributed peer review (DPR) systems, to allocate micro-grants for the fast prototyping of innovative solutions. Here we describe and evaluate the implementation of this community review across 147 projects from the “Just One Giant Lab’s OpenCOVID19 initiative” and “Helpful Engineering” open research communities. The community review process uses granular review forms and requires the participation of grant applicants in the review process. We show that this system is fast, with a median duration of 10 days, scalable, with a median of 4 reviewers per project independent of the total number of projects, and fair, with project rankings highly preserved after the synthetic removal of reviewers. We investigate potential bias introduced by involving applicants in the process, and find that review scores from both applicants and non-applicants have a similar correlation of r=0.28 with other reviews within a project, matching previous observations using traditional approaches. Finally, we find that the ability of projects to apply to several rounds allows to both foster the further implementation of successful early prototypes, as well as provide a pathway to constructively improve an initially failing proposal in an agile manner. This study quantitatively highlights the benefits of a frugal community review system for agile resource allocation

Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition.

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell

A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity

Proteomic, localization, and enzymatic activity screens in fission yeast reveal how deubiquitinating enzyme localization and function are tuned

Differential GAP requirement for Cdc42-GTP polarization during proliferation and sexual reproduction.

The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPase-activating protein (GAP) in the fission yeast Schizosaccharomyces pombe Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues

Inhibition of Ras activity coordinates cell fusion with cell-cell contact during yeast mating.

In the fission yeast Schizosaccharomyces pombe, pheromone signaling engages a signaling pathway composed of a G protein-coupled receptor, Ras, and a mitogen-activated protein kinase (MAPK) cascade that triggers sexual differentiation and gamete fusion. Cell-cell fusion requires local cell wall digestion, which relies on an initially dynamic actin fusion focus that becomes stabilized upon local enrichment of the signaling cascade on the structure. We constructed a live-reporter of active Ras1 (Ras1-guanosine triphosphate [GTP]) that shows Ras activity at polarity sites peaking on the fusion structure before fusion. Remarkably, constitutive Ras1 activation promoted fusion focus stabilization and fusion attempts irrespective of cell pairing, leading to cell lysis. Ras1 activity was restricted by the guanosine triphosphatase-activating protein Gap1, which was itself recruited to sites of Ras1-GTP and was essential to block untimely fusion attempts. We propose that negative feedback control of Ras activity restrains the MAPK signal and couples fusion with cell-cell engagement

Kriterien für die Akzeptanz von recyceltem Phosphatdünger aus Abwasser und Klärschlamm im ökologischen Landbau

Plant available phosphorus is a limiting factor in organic farming. In the wake of an increasing awareness of the planetary boundaries of phosphate rock deposits, various research efforts to develop fertilizers based on recycled phosphorus from wastewater, sewage sludge and sewage sludge ashes have been initiated. The study aimed at the future potential of these fertilizers for organic farming and tried to identify and evaluate criteria that need to be fulfilled so that fertilizers of recycled phosphorus would be accepted by organic farmers. The survey revealed that the most important criterion for the acceptance is the non-existence of hazardous substances in the fertilizer. Plant availability, low energy input and high transparency of the production process of the fertilizers were also regarded as relatively important. Spreadability, price, public acceptance and superficial distinction compared to mineral fertilizers play a minor role for the acceptance