The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation.

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

On the Evolution of Specificity in Members of the Yeast Amino Acid Transporter Family as Parts of Specific Metabolic Pathways.

In the recent years, molecular modeling and substrate docking, coupled with biochemical and genetic analyses have identified the substrate-binding residues of several amino acid transporters of the yeast amino acid transporter (YAT) family. These consist of (a) residues conserved across YATs that interact with the invariable part of amino acid substrates and (b) variable residues that interact with the side chain of the amino acid substrate and thus define specificity. Secondary structure sequence alignments showed that the positions of these residues are conserved across YATs and could thus be used to predict the specificity of YATs. Here, we discuss the potential of combining molecular modeling and structural alignments with intra-species phylogenetic comparisons of transporters, in order to predict the function of uncharacterized members of the family. We additionally define some orphan branches which include transporters with potentially novel, and to be characterized specificities. In addition, we discuss the particular case of the highly specific l-proline transporter, PrnB, of Aspergillus nidulans, whose gene is part of a cluster of genes required for the utilization of proline as a carbon and/or nitrogen source. This clustering correlates with transcriptional regulation of these genes, potentially leading to the efficient coordination of the uptake of externally provided l-Pro via PrnB and its enzymatic degradation in the cell.

The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters.

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly(56), Thr(57)), TMS3 (Glu(138)), and TMS6 (Phe(248)), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle.

Amphiphilic Chlorin-ß-cyclodextrin Conjugates in Photo-Triggered Drug Delivery: The Role of Aggregation.

Conjugates of chlorins with ß-cyclodextrin connected either directly or via a flexible linker were prepared. In aqueous medium these amphiphilic conjugates were photostable, produced singlet oxygen at a rate similar to clinically used temoporfin and formed irregular nanoparticles via aggregation. Successful loading with the chemotherapeutic drug tamoxifen was evidenced in solution by the UV-Vis spectral changes and dynamic light scattering profiles. Incubation of MCF-7 cells with the conjugates revealed intense spotted intracellular fluorescence suggestive of accumulation in endosome/lysosome compartments, and no dark toxicity. Incubation with the tamoxifen-loaded conjugates revealed also practically no dark toxicity. Irradiation of cells incubated with empty conjugates at 640 nm and 4.18 J/cm2 light fluence caused >50 % cell viability reduction. Irradiation following incubation with tamoxifen-loaded conjugates resulted in even higher toxicity (74 %) indicating that the produced reactive oxygen species had triggered tamoxifen release in a photochemical internalization (PCI) mechanism. The chlorin-ß-cyclodextrin conjugates displayed less-lasting effects with time, compared to the corresponding porphyrin-ß-cyclodextrin conjugates, possibly due to lower tamoxifen loading of their aggregates and/or their less effective lodging in the cell compartments' membranes. The results suggest that further to favorable photophysical properties, other parameters are important for the in vitro effectiveness of the photodynamic systems.

Eisosome distribution and localization in the meiotic progeny of Aspergillus nidulans.

In the model filamentous fungus Aspergillus nidulans, PilA and PilB, two homologues of the Saccharomyces cerevisiae eisosome proteins Pil1/Lsp1, and SurG, a strict orthologue of Sur7, are assembled and form tightly packed structures in conidiospores. As A. nidulans differs in its reproduction pattern from the Saccharomycotina in that it has the ability to reproduce through two different types of spores, conidiospores and ascospores, the products of the asexual and the sexual cycle respectively, we investigated the eisosome distribution and localization during the sexual cycle. Our results show that core eisosome proteins PilA, PilB and SurG are not expressed in hülle cells or early ascospores, but are expressed in mature ascospores. All eisosomal proteins form punctate structures at the membrane of late ascospores. In mature but quiescent ascospores, PilA forms static punctate structures at the plasma membrane. PilB also was observed at the ascospore membrane as well, with higher concentration at the areas where the two halves of ascospores are joined together. Finally, SurG was localized both at the membrane of ascospores and perinuclearly. In germlings originating from ascospores the punctate structures were shown to be composed only of PilA. PilB is diffused in the cytoplasm and SurG was located in vacuoles and endosomes. This altered localization is identical to that found in germlings originated from conidiospores. In germinated ascospores PilA foci did not colocalise with the highly mobile and transient peripheral punctate structures of AbpA, a marker for sites of clathrin-mediated endocytosis. Deletions of each one or all the three core eisosomal genes do not affect viability or germination of ascospores. In the presence of myriocin - a specific inhibitor of sphingolipid biosynthesis - PilA-GFP foci of ascospore germlings were less numerous and their distribution was significantly altered, suggesting a correlation between PilA foci and sphingolipid biosynthesis.

Small anticancer drug release by light: Photochemical internalization of porphyrin-ß-cyclodextrin nanoparticles.

Aiming to engineer simple, neutral, strongly amphiphilic photoactive nanoparticles (NPs) to specifically target cancer cell lysosomes for drug transport and light-controlled release, new conjugates of ß-cyclodextrin with highly hydrophobic triphenylporphyrin bearing different alkyl chains, were synthesized. Although differently sized, all conjugates self-assemble into ~60 nm NPs in water and display similar photoactivity. The NPs target selectively the lysosomes of breast adenocarcinoma MCF-7 cells, embedding in vesicular membranes, as experiments with model liposomes indicate. Either empty or drug-loaded, the NPs lack dark toxicity for 48 h. They bind with differently structured anticancer drugs tamoxifen and gemcitabine as its N-adamantyl derivative. Red light irradiation of cells incubated with drug-loaded NPs results in major reduction of viability (>85 %) for 48 h displaying significant synergy of photo-chemotoxicity, as opposed to empty NPs, and to loaded non-irradiated NPs, in manifestation of photochemical internalization (PCI). Our approach expands the field of PCI into different small molecule chemotherapeutics.

Ferroptosis-protective membrane domains in quiescence.

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-ß-cyclodextrin as sequestrant and cell uptake modulator.

In the search for photosensitizers with chemical handles to facilitate their integration into complex drug delivery nanosystems, new, unsymmetrically substituted, water insoluble meso-tetraphenylporphyrin and meso-tetra(m-hydroxyphenyl)porphyrin derivatives bearing one carboxyalkyl side chain were synthesized. Permethyl-ß-cyclodextrin (pMßCD) was their ideal monomerizing host and highly efficient shuttle to transfer them into water. New assembly modes of the extremely stable (Kbinding > 1012 M-2) 2:1 complexes were identified. The complexes are photostable and do not disassemble in FBS-containing cell culture media for 24 h. Incubation of breast cancer MCF-7 cells with the complexes results in intense intracellular fluorescence, strongly enhanced in the endoplasmic reticulum (ER), high photokilling efficiency (~90%) and low dark toxicity. pMßCD stands out as a very capable molecular isolator of mono-carboxyalkyl-arylporphyrins that increases uptake and modulates their localization in the cells. The most efficient porphyrins are envisaged as suitable photosensitizers that can be linked to biocompatible drug carriers for photo- and chemo-therapy applications.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software.

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

A highly conserved mechanism for the detoxification and assimilation of the toxic phytoproduct L-azetidine-2-carboxylic acid in Aspergillus nidulans.

Plants produce toxic secondary metabolites as defense mechanisms against phytopathogenic microorganisms and predators. L-azetidine-2-carboxylic acid (AZC), a toxic proline analogue produced by members of the Liliaceae and Agavaciae families, is part of such a mechanism. AZC causes a broad range of toxic, inflammatory and degenerative abnormalities in human and animal cells, while it is known that some microorganisms have evolved specialized strategies for AZC resistance. However, the mechanisms underlying these processes are poorly understood. Here, we identify a widespread mechanism for AZC resistance in fungi. We show that the filamentous ascomycete Aspergillus nidulans is able to not only resist AZC toxicity but also utilize it as a nitrogen source via GABA catabolism and the action of the AzhA hydrolase, a member of a large superfamily of detoxifying enzymes, the haloacid dehalogenase-like hydrolase (HAD) superfamily. This detoxification process is further assisted by the NgnA acetyltransferase, orthologue of Mpr1 of Saccharomyces cerevisiae. We additionally show that heterologous expression of AzhA protein can complement the AZC sensitivity of S. cerevisiae. Furthermore, a detailed phylogenetic analysis of AzhA homologues in Fungi, Archaea and Bacteria is provided. Overall, our results unravel a widespread mechanism for AZC resistance among microorganisms, including important human and plant pathogens.

Engineering Mitochondriotropic Carbon Dots for Targeting Cancer Cells.

Aiming to understand and enhance the capacity of carbon dots (CDs) to transport through cell membranes and target subcellular organelles-in particular, mitochondria-a series of nitrogen-doped CDs were prepared by the one-step microwave-assisted pyrolysis of citric acid and ethylenediamine. Following optimization of the reaction conditions for maximum fluorescence, functionalization at various degrees with alkylated triphenylphosphonium functional groups of two different alkyl chain lengths afforded a series of functionalized CDs that exhibited either lysosome or mitochondria subcellular localization. Further functionalization with rhodamine B enabled enhanced fluorescence imaging capabilities in the visible spectrum and allowed the use of low quantities of CDs in relevant experiments. It was thus possible, by the appropriate selection of the alkyl chain length and degree of functionalization, to attain successful mitochondrial targeting, while preserving non-toxicity and biocompatibility. In vitro cell experiments performed on normal as well as cancer cell lines proved their non-cytotoxic character and imaging potential, even at very low concentrations, by fluorescence microscopy. Precise targeting of mitochondria is feasible with carefully designed CDs that, furthermore, are specifically internalized in cells and cell mitochondria of high transmembrane potential and thus exhibit selective uptake in malignant cells compared to normal cells.

Assessment of the operation of a pilot program of preventive medicine for adults in three primary care centers of Athens and Piraeus: a cross-sectional study.

OBJECTIVE: To evaluate the operation of the pilot Program of Preventive Medicine for Adults (PPMA) from the provision of healthcare services in three primary health care centers of Athens and Piraeus (Greece). DESIGN: An observational, cross-sectional study was undertaken. A structured questionnaire was used to collect data. SETTING: Three public primary health centers of Athens, Greece. PARTICIPANTS: 142 participants, 40-55 years of age, with no previously identified/diagnosed health issues (overall healthy) that should not have undergone any type of screening in the past 12 months. MEASURES AND RESULTS: The majority of participants were female (75.4%), aged 40-45 years (43.7%), Greek nationals (88%), employed (62.7%) and high school graduates (52.8%). 68.3% of participants scheduled an appointment in the first two days and 58.9% waited up to 5 minutes in the waiting room. The paramedical personnel scored higher evaluation (4.94) than the doctor (4.61), but all health care centers received a very high general evaluation (4.87). Multiple linear regression showed significant association between the evaluation of the pilot PPMA, the evaluation of health visitor/nurse (B = .240) and the communication of results to participants (B = .245). CONCLUSIONS: Findings show an extremely positive evaluation of the pilot PPMA, a result that can be used by healthcare managers for the expansion of the screening program to the general population.

Fungal plasma membrane domains.

The plasma membrane (PM) performs a plethora of physiological processes, the coordination of which requires spatial and temporal organization into specialized domains of different sizes, stability, protein/lipid composition and overall architecture. Compartmentalization of the PM has been particularly well studied in the yeast Saccharomyces cerevisiae, where five non-overlapping domains have been described: The Membrane Compartments containing the arginine permease Can1 (MCC), the H+-ATPase Pma1 (MCP), the TORC2 kinase (MCT), the sterol transporters Ltc3/4 (MCL), and the cell wall stress mechanosensor Wsc1 (MCW). Additional cortical foci at the fungal PM are the sites where clathrin-dependent endocytosis occurs, the sites where the external pH sensing complex PAL/Rim localizes, and sterol-rich domains found in apically grown regions of fungal membranes. In this review, we summarize knowledge from several fungal species regarding the organization of the lateral PM segregation. We discuss the mechanisms of formation of these domains, and the mechanisms of partitioning of proteins there. Finally, we discuss the physiological roles of the best-known membrane compartments, including the regulation of membrane and cell wall homeostasis, apical growth of fungal cells and the newly emerging role of MCCs as starvation-protective membrane domains.

Characterization of AnNce102 and its role in eisosome stability and sphingolipid biosynthesis.

The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid biosynthesis, since their deletions partially suppress the thermosensitivity of basA mutant encoding sphingolipid C4-hydroxylase and the growth defects observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or pharmacological depletion of sphingolipids, conditions that induce the production of Reactive Oxygen Species (ROS), and can be partially overcome by deletion of pilA and/or annce102 at high temperatures. Consistent with these findings, pilAΔ and annce102Δ also show differential sensitivity to various oxidative agents, while AnNce102 overexpression can bypass sphingolipid depletion regarding the PilA/SurG foci number and organization, also leading to the mislocalization of PilA to septa.