continuous-high-resolution-microscopic-observation-replicative-aging

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

ETH Zurich

We formulate a U(1) gauge theory of the Hubbard model in the slave-rotor representation. From this formalism it is argued that spin liquid phases may exist near the Mott transition in the Hubbard model on triangular and honeycomb lattices at half filling. The organic compound kappa-(BEDT-TTF)2Cu2(CN)3 is a good candidate for the spin liquid state on a triangular lattice. We predict a highly unusual temperature dependence for the thermal conductivity of this material.

U(1) gauge theory of the Hubbard model: spin liquid states and possible application to kappa-(BEDT-TTF)2Cu2(CN)3.

Recent experiments on the organic compound kappa-(BEDT-TTF)2Cu2(CN)3 raise the possibility that the system may be described as a quantum spin liquid. Here we propose a pairing state caused by the "Amperean" attractive interaction between spinons on a Fermi surface mediated by the U(1) gauge field. We show that this state can explain many of the observed low temperature phenomena and discuss testable consequences.

Amperean pairing instability in the U1 spin liquid state with fermi surface and application to kappa-(BEDT-TTF)2Cu2(CN)3.

We calculate and compare the effects of aprotic vs protic solvent on the rate of SN2 reaction [F- + C3H7OMs--> C3H7F + OMs-]. We find that aprotic solvent acetonitrile is more efficient than a small protic solvent such as methanol. Bulky protic solvent (tert-butyl alcohol) is predicted to be quite efficient, giving the rate constant that is similar to that in CH3CN. Our calculated relative activation barriers of the SN2 reaction in methanol, tert-butyl alcohol, and CH3CN are in good agreement with experimental observations.

Efficiency of bulky protic solvent for SN2 reaction.

We discuss the implications of approximate particle-hole symmetry in a half-filled Landau level in which a paired quantum Hall state forms. We note that the Pfaffian state is not particle-hole symmetric. Therefore, in the limit of vanishing Landau-level mixing, in which particle-hole transformation is an exact symmetry, the Pfaffian spontaneously breaks this symmetry. There is a particle-hole conjugate state, which we call the anti-Pfaffian, which is degenerate with the Pfaffian in this limit. We observe that strong Landau-level mixing should favor the Pfaffian, but it is an open problem which state is favored for the moderate Landau-level mixing which is present in experiments. We discuss the bulk and edge physics of the anti-Pfaffian. We analyze a simplified model in which transitions between analogs of the two states can be studied in detail. Finally, we discuss experimental implications.

Particle-hole symmetry and the nu=5/2 quantum Hall state.

We propose a many-body generalization of the Z2 topological invariant for the quantum spin Hall insulator, which does not rely on single-particle band structures. The invariant is derived as a topological obstruction that distinguishes topologically distinct many-body ground states on a torus. It is also expressed as a Wilson loop of the SU(2) Berry gauge field, which is quantized due to time-reversal symmetry.

Many-body generalization of the Z2 topological invariant for the quantum spin Hall effect.

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R(2)=0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively. In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n=253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea.

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 beta-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.

Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.

Particle focusing in planar geometries is essentially required in order to develop cost-effective lab-on-a-chips, such as cell counting and point-of-care (POC) devices. In this study, a novel method for sheathless particle focusing, called "Elasto-Inertial Particle Focusing", was demonstrated in a straight microchannel. The particles were notably aligned along the centerline of the straight channel under a pressure-driven flow without any additional external force or apparatus after the addition of an elasticity enhancer: PEO (poly(ethylene oxide)) (âˆ¼O(100) ppm). As theoretically predicted (elasticity number: Elâ‰ˆO(100)), multiple equilibrium positions (centerline and corners) were observed for the viscoelastic flow without inertia, whereas three-dimensional particle focusing only occurred when neither the elasticity nor the inertia was negligible. Therefore, the three-dimensional particle focusing mechanism was attributed to the synergetic combination of the elasticity and the inertia (elasticity number: Elâ‰ˆO(1-10)). Furthermore, from the size dependence of the elastic force upon particles, we demonstrated that a mixture of 5.9 and 2.4 Âµm particles was separated at the exit of the channel in viscoelastic flows. We expect that this method can contribute to develop the miniaturized flow cytometry and microdevices for cell and particle manipulation.

Sheathless elasto-inertial particle focusing and continuous separation in a straight rectangular microchannel.

We have developed a single-cell assay platform that allows quantitative analysis of single cell chemotaxis by dynamic morphogenetic gradients, subcellular microscopic imaging and automated image analysis, and have applied these to measure cellular polarization of budding yeast. The computer-controlled microfluidic device regulates the gradient profile at any given time, and allows quantitative monitoring of cell morphology and the localization and expression of specific marker proteins during the dynamic polarization process. With this integrated experimental system, we compare the polarized signaling response of wild-type and far1-H7 mutant cells, which express a truncated Far1 protein unable to interact with Cdc24. Our results confirm that Far1 functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling. Moreover, by changing the gradient profile and estimating the number of bound surface receptors, we quantitatively address why surprisingly small differences in pheromone concentration across yeast cells can be amplified into a robust polarity axis. This integrated single cell experimental platform thus opens the possibility to quantitatively investigate the molecular regulatory mechanism of chemotaxis in yeast, which serves as a paradigm to understand the fundamental processes involved in cancer metastasis, angiogenesis and axon generation.

Quantitative and dynamic assay of single cell chemotaxis.

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform.

Much difficulty has been encountered in manipulating small-scale materials, such as submicron colloidal particles and macromolecules (e.g., DNA and proteins), in microfluidic devices since diffusion processes due to thermal (Brownian) motion become more pronounced with decreasing particle size. Here, we present a novel approach for the continuous focusing of such small-scale materials. First, we successfully focused fluorescent submicron polystyrene (PS) beads along equilibrium positions in microchannels through the addition of a small amount water-soluble polymer [500 ppm poly(ethylene oxide) (PEO)]. Lateral migration velocity significantly depends upon the viscoelastic effect (Weissenberg number: Wi) and the aspect ratio of particle size to channel height (a/h). Interestingly, focusing using viscoelastic flows was also observed for flexible DNA molecules (Î»-DNA and T4-DNA), which have radii of gyration (R(g)) of approximately 0.69 Î¼m and 1.5 Î¼m, respectively. This small-scale material manipulation using medium viscoelasticity will contribute to the design of nanoparticle separation and genomic mapping devices.

Lateral migration and focusing of colloidal particles and DNA molecules under viscoelastic flow.

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy.

Microscopy can provide invaluable information about biological processes at the single cell level. It remains a challenge, however, to extract quantitative information from these types of datasets. We have developed an image analysis platform named YeastQuant to simplify data extraction by offering an integrated method to turn time-lapse movies into single cell measurements. This platform is based on a database with a graphical user interface where the users can describe their experiments. The database is connected to the engineering software Matlab, which allows extracting the desired information by automatically segmenting and quantifying the microscopy images. We implemented three different segmentation methods that recognize individual cells under different conditions, and integrated image analysis protocols that allow measuring and analyzing distinct cellular readouts. To illustrate the power and versatility of YeastQuant, we investigated dynamic signal transduction processes in yeast. First, we quantified the expression of fluorescent reporters induced by osmotic stress to study noise in gene expression. Second, we analyzed the dynamic relocation of endogenous proteins from the cytoplasm to the cell nucleus, which provides a fast measure of pathway activity. These examples demonstrate that YeastQuant provides a versatile and expandable database and an experimental framework that improves image analysis and quantification of diverse microscopy-based readouts. Such dynamic single cell measurements are highly needed to establish mathematical models of signal transduction pathways.

An integrated image analysis platform to quantify signal transduction in single cells.

We present an efficient method for measuring cell stretching based on three-dimensional viscoelastic particle focusing. We suspended cells in a biocompatible viscoelastic medium [poly(vinylpyrrolidone) solution in phosphate-buffered saline]. The medium viscoelasticity significantly homogenized the trajectories of cells along the centerline of a simple straight channel, which could not be achieved in conventional Newtonian media. More than 95% of red blood cells (RBCs) were successfully delivered to the stagnation point of a cross-slot microchannel and stretched by extensional flow. By computational simulations, we proved that this method prevents inaccuracies due to random lateral distributions of cells and, further, guarantees rotational-free cell stretching along the shear-free channel centerline. As a demonstration, we characterized the differences in RBC deformabilities among various heat treatments. Furthermore, we monitored the decrease of deformability due to nutrient starvation in human mesenchymal stem cells. We envisage that our novel method can be extended to versatile applications such as the detection of pathophysiological evolution in impaired RBCs due to malaria or diabetes and the monitoring of cell quality in stem cell therapeutics.

Cell stretching measurement utilizing viscoelastic particle focusing.

This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50-100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ~7 h.

Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast.

Plant leaf cuticle is related to the prevention of moisture loss, transpiration, and diffusion of light reflection. The purpose of this study was to examine the morphological characteristics of ginseng leaves in ginseng plants resistant and susceptible to high temperature injury (HTI) to be related with the leaf-burning. For the HTI resistant lines Yunpoong, high-temperature injury resistance (HTIR) 1, HTIR 2, and HTIR 3, and the HTI-susceptible line Chunpoong, the cuticle densities were 53.0%, 46.2%, 44.9%, 48.0%, and 17.0%; the adaxial leaf cuticle layers were 141.3, 119.7, 119.7, 159.4, and 85.0 nm in thickness; the abaxial leaf cuticle layers were 153.6, 165.8, 157.9, 199.6, and 119.4 nm in thickness; and the stomtal lengths were 21.7, 32.4, 29.4, 30.9, and 21.8 Î¼m, respectively. All of these aspects suggest that HTI resistant lines have higher cuticle density, thicker adaxial and abaxial leaf cuticle layers, and longer of stomta length than the HTI-susceptible line, protecting leaves from moisture loss and excessive transpiration under high temperatures to be resistant against the leaf-burning.

Article	Year
U(1) gauge theory of the Hubbard model: spin liquid states and possible application to kappa-(BEDT-TTF)2Cu2(CN)3. Physical review letters\| PubMed ID: 16090761	2005
Amperean pairing instability in the U1 spin liquid state with fermi surface and application to kappa-(BEDT-TTF)2Cu2(CN)3. Physical review letters\| PubMed ID: 17358978	2007
Efficiency of bulky protic solvent for SN2 reaction. Organic letters\| PubMed ID: 18052384	2008
Particle-hole symmetry and the nu=5/2 quantum Hall state. Physical review letters\| PubMed ID: 18233397	2007
Many-body generalization of the Z2 topological invariant for the quantum spin Hall effect. Physical review letters\| PubMed ID: 18518407	2008
Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea. Research in veterinary science\| PubMed ID: 20060551	2010
Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase. The EMBO journal\| PubMed ID: 20581803	2010
Sheathless elasto-inertial particle focusing and continuous separation in a straight rectangular microchannel. Lab on a chip\| PubMed ID: 20976348	2011
Quantitative and dynamic assay of single cell chemotaxis. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 22230969	2012
Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22421136	2012
Lateral migration and focusing of colloidal particles and DNA molecules under viscoelastic flow. Lab on a chip\| PubMed ID: 22776909	2012
Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. The EMBO journal\| PubMed ID: 22885598	2012
An integrated image analysis platform to quantify signal transduction in single cells. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 22976484	2012
Cell stretching measurement utilizing viscoelastic particle focusing. Analytical chemistry\| PubMed ID: 23163397	2012
Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast. Nature protocols\| PubMed ID: 23640166	2013
Morphological Characteristics of Ginseng Leaves in High-Temperature Injury Resistant and Susceptible Lines of Panax ginseng Meyer. Journal of ginseng research\| PubMed ID: 23717091	2011

Sung Sik Lee

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