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The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing.

Central to proteopathies and leading to most age-related neurodegenerative disorders is a failure in protein quality control (PQC). To harness the toxicity of misfolded and damaged disease proteins, such proteins are either refolded, degraded by temporal PQC, or sequestered by spatial PQC into specific, organelle-associated, compartments within the cell. Here, we discuss the impact of vesicle trafficking pathways in general, and syntaxin 5 in particular, as key players in spatial PQC directing misfolded proteins to the surface of vacuole and mitochondria, which facilitates their clearance and detoxification. Since boosting vesicle trafficking genetically can positively impact on spatial PQC and make cells less sensitive to misfolded disease proteins, we speculate that regulators of such trafficking might serve as therapeutic targets for age-related neurological disorders.

Coronavirus family has caused several human illnesses, the latest caused by SARS-CoV-2, has led to COVID-19 pandemic posing serious threat to global health. A SARS-CoV-2 variant encoding a D614G mutation in the viral spike (S) protein has now become the most prevalent form of the virus worldwide, suggesting a fitness advantage for the mutant. The G614 variant is associated with higher upper respiratory tract viral load, higher infectivity, increased total S protein incorporation into the virion, reduced S1 shedding and a conformational change leading to a more ACE2- binding and fusion- competent state. However, it does not seem to be correlated to increased disease severity or escape neutralizing antibodies.

Coronaviruses primarily cause zoonotic infections, however in the past few decades several interspecies transmissions have occurred, the last one by SARS-CoV-2, causing COVID-19 pandemic, posing serious threat to global health. The SARS-CoV-2 spike (S) protein plays an important role in viral attachment, fusion and entry. However, other structural and non-structural SARS-CoV-2 proteins are potential influencers in virus pathogenicity. Among these proteins; Orf3, Orf8, and Orf10 show the least homology to SARSCoV proteins and therefore should be further studied for their abilities to modulate antiviral and inflammatory responses. Here, we discuss how SARS-COV-2 interacts with our immune system.

Arsenite is widely present in nature; therefore, cells have evolved mechanisms to prevent arsenite influx and promote efflux. In yeast (Saccharomyces cerevisiae), the aquaglyceroporin Fps1 mediates arsenite influx and efflux. The mitogen-activated protein kinase (MAPK) Hog1 has previously been shown to restrict arsenite influx through Fps1. In this study, we show that another MAPK, Slt2, is transiently phosphorylated in response to arsenite influx. Our findings indicate that the protein kinase activity of Slt2 is required for its role in arsenite tolerance. While Hog1 prevents arsenite influx via phosphorylation of T231 at the N-terminal domain of Fps1, Slt2 promotes arsenite efflux through phosphorylation of S537 at the C terminus. Our data suggest that Slt2 physically interacts with Fps1 and that this interaction depends on phosphorylation of S537. We hypothesize that Hog1 and Slt2 may affect each other's binding to Fps1, thereby controlling the opening and closing of the channel.

Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in treating numerous ailments and diseases. Hence, inventing strategies to tune cellular arsenic uptake based on purpose is striking. Here, we describe an approach in which the arsenite uptake can be increased using a MAPK inhibitor. Employing microfluidic flow chambers in combination with optical tweezers and fluorescent microscopy, we elevated the influx of arsenite into the yeast Saccharomyces cerevisiae cells following short-term treatment with a Hog1 kinase inhibitor. The increase in arsenite uptake was followed on arsenite triggered redistribution of a reporter protein, Hsp104-GFP, which was imaged over time. The effect was even more pronounced when the yeast mother and daughter cells were analyzed disjointedly, an opportunity provided owing to single-cell analysis. Our data firstly provide a strategy to increase arsenite uptake and secondly show that arsenite triggered aggregates, previously shown to be sites of damaged proteins, are distributed asymmetrically and less accumulated in daughter cells. Inventing approaches to tune arsenite uptake has a great value for its use in environmental as well as medical applications.

The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS) using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells) with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III)) uptake in yeast. Redistribution of a green fluorescent protein (GFP)-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min−1, moderate = 50 nL min−1, and high = 100 nL min−1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.

Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.

Single cell analysis techniques provide a unique opportunity of determining the intercellular heterogeneity in a cell population, which due to genotype variations and different physiological states of the cells i.e. size, shape and age, cannot be retrieved from averaged cell population values. In order to obtain high-value quantitative data from single-cell experiments it is important to have experimental platforms enabling high-throughput studies. Here, we present a microfluidic chip, which is capable of capturing individual cells in suspension inside separate traps. The device consists of three adjacent microchannels with separate inlets and outlets, laterally connected through the V-shaped traps. Vshaped traps, with openings smaller than the size of a single cell, are fabricated in the middle (main) channel perpendicular to the flow direction. Cells are guided into the wells by streamlines of the flows and are kept still at the bottom of the traps. Cells can then be exposed to extracellular stimuli either in the main or the side channels. Microchannels and traps of different sizes can be fabricated in polydimethylsiloxane (PDMS), offering the possibility of independent studies on cellular responses with different cell types and different extracellular environmental changes. We believe that this versatile high-throughput cell trapping approach will contribute to further development of the current knowledge and information acquired from single-cell studies and provide valuable statistical experimental data required for systems biology. © (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Investigating the behavior of single cells in a high-throughput manner calls for the design and realization of innovative, robust and versatile devices capable of conducting adaptable experiments to address the desired biological questions.For instance, the possibility of running experiments that compare the reaction of different cell types to a specific stimulus or the effect of a given substance at different concentrations on a specific cell type is highly desirable. One inherent challenge associated with running such experiments, however, is the existence of random uncertainty factors from experiment to experiment. Examples of this could be common measurement errors in preparing exact substance concentrations or the likeness of consecutive cell cultures.Here, we report the design and fabrication of a high-throughput microfluidic platform intended for parallel investigations on single cells upon extracellular environmental changes.We fabricated a hydrodynamic-based cell isolation microfluidic block in polydimethylsiloxane (PDMS) with two secluded entities. The comb-shaped trapping zone in each entity consists of 52 V-formed individual traps. Each trap has the dimensions of 10 μm × 10 μm and connects two complement microchannels via a 2-μm wide confinement opening. After the cell-loading step, isolated cells can undergo several chemical-rinsing steps and, in combination with fluorescence microscopy imaging, cellular response can be read and analyzed.This easy-to-operate, parallel microfluidic platform enables the simultaneous comparison of the read out between two portions of a particular cell passage to various chemical stresses or different concentrations of one specific stimulus.Moreover cellular responses from two different cell types to a known stress substance can be followed over time.We foresee that this device can be considered as the first building block of a completely integrated lab-on-a-chip platform for concentration-dependent drug screening applications and comparative single-cell data acquisition.

Cellular signalling networks integrate environmental stimuli with the information on cellular status. These networks must be robust against stochastic fluctuations in stimuli as well as in the amounts of signalling components. Here, we challenge the yeast HOG signal-transduction pathway with systematic perturbations in components' expression levels under various external conditions in search for nodes of fragility. We observe a substantially higher frequency of fragile nodes in this signal-transduction pathway than that has been observed for other cellular processes. These fragilities disperse without any clear pattern over biochemical functions or location in pathway topology and they are largely independent of pathway activation by external stimuli. However, the strongest toxicities are caused by pathway hyperactivation. In silico analysis highlights the impact of model structure on in silico robustness, and suggests complex formation and scaffolding as important contributors to the observed fragility patterns. Thus, in vivo robustness data can be used to discriminate and improve mathematical models.

Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1Δ sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.

Regulating arsenic uptake is imperative due to its carcinogenicity. Combining microfluidics, optical tweezers and fluorescence microscopy, the arsenite uptake by Fps1 using a selective kinase inhibitor is investigated using a single cell analysis platform.