scholarly journals Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

2020 ◽  
Author(s):  
Santosh Adhikari ◽  
Joe Moscatelli ◽  
Elias M. Puchner
Keyword(s):  
Stationary Phase ◽  
Single Molecule ◽  
Lipid Droplets ◽  
Fold Increase ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Yeast Cells ◽  
Lipid Homeostasis ◽  
Localization Microscopy ◽  
Live Yeast

AbstractLipid droplets (LDs) are dynamic lipid storage organelles needed for lipid homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs, as well as enzymes required for LD growth and turnover. Due to LD size below the optical diffraction limit, bulk fluorescence microscopy cannot observe the density and dynamics of specific LD enzymes. Here, we employ quantitative photo-activated localization microscopy (PALM) to study the density of the fatty acid activating protein Faa4 on LDs during log, stationary and lag phases in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where the highest Faa4 densities are measured. During transition to the stationary phase LDs translocate to the vacuolar surface and lumen with a ~2-fold increased surface area and a ~2.5-fold increase in Faa4 density, suggesting its role in LD expansion. The increased Faa4 density on LDs is caused by its ~5-fold increased expression level. When lipolysis is induced in stationary-phase cells by diluting them for 2 hrs in fresh medium, Faa4 shuttles to the vacuole through the two observed routes of ER- and lipophagy. The observed vacuolar localization of Faa4 may help activating fatty acids for membrane expansion and reduces Faa4 expression to levels found in the log phase.

scholarly journals Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

2021 ◽  
pp. mbc.E20-11-0695
Author(s):  
Santosh Adhikari ◽  
Joe Moscatelli ◽  
Elias M. Puchner
Keyword(s):  
Fatty Acid ◽  
Single Molecule ◽  
Lipid Droplets ◽  
Fold Increase ◽  
Yeast Cells ◽  
Exogenous Fatty Acid ◽  
Localization Microscopy ◽  
Fatty Acid Chain ◽  
Chain Lengths ◽  
Live Yeast

Lipid droplets (LDs) are dynamic organelles for lipid storage and homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs and enzymes required for LD growth and turnover. The small size of LDs precludes the observation of their associated enzyme densities and dynamics with conventional fluorescence microscopy. Here, we employ quantitative photo-activated localization microscopy to study the density of the fatty acid activating enzyme Faa4 on LDs in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where their emergence and expansion is mediated by the highest observed Faa4 densities. During transition to the stationary phase LDs with a ∼2-fold increased surface area translocate to the vacuolar surface and lumen and exhibit a ∼2.5-fold increase in Faa4 density. The increased Faa4 density on LDs further suggests its role in LD expansion, is caused by its ∼5-fold increased expression level and is specific to exogenous fatty acid chain-lengths. When lipolysis is induced by refreshed medium, Faa4 shuttles through ER- and lipophagy to the vacuole, where it may activate fatty acids for membrane expansion and degrade to reset cellular Faa4 abundance to levels in the log phase. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals Efficient Cross-Correlation Filtering of One- and Two-Color Single Molecule Localization Microscopy Data

2021 ◽  
Vol 1 ◽  
Author(s):  
Angel Mancebo ◽  
Dushyant Mehra ◽  
Chiranjib Banerjee ◽  
Do-Hyung Kim ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Cross Correlation ◽  
Pair Correlation ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Data Sets ◽  
Oligomeric State ◽  
Localization Microscopy ◽  
Cross Correlation Analysis ◽  
Single Molecule Localization Microscopy

Single molecule localization microscopy has become a prominent technique to quantitatively study biological processes below the optical diffraction limit. By fitting the intensity profile of single sparsely activated fluorophores, which are often attached to a specific biomolecule within a cell, the locations of all imaged fluorophores are obtained with ∼20 nm resolution in the form of a coordinate table. While rendered super-resolution images reveal structural features of intracellular structures below the optical diffraction limit, the ability to further analyze the molecular coordinates presents opportunities to gain additional quantitative insights into the spatial distribution of a biomolecule of interest. For instance, pair-correlation or radial distribution functions are employed as a measure of clustering, and cross-correlation analysis reveals the colocalization of two biomolecules in two-color SMLM data. Here, we present an efficient filtering method for SMLM data sets based on pair- or cross-correlation to isolate localizations that are clustered or appear in proximity to a second set of localizations in two-color SMLM data. In this way, clustered or colocalized localizations can be separately rendered and analyzed to compare other molecular properties to the remaining localizations, such as their oligomeric state or mobility in live cell experiments. Current matrix-based cross-correlation analyses of large data sets quickly reach the limitations of computer memory due to the space complexity of constructing the distance matrices. Our approach leverages k-dimensional trees to efficiently perform range searches, which dramatically reduces memory needs and the time for the analysis. We demonstrate the versatile applications of this method with simulated data sets as well as examples of two-color SMLM data. The provided MATLAB code and its description can be integrated into existing localization analysis packages and provides a useful resource to analyze SMLM data with new detail.

scholarly journals Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

2021 ◽  
Author(s):  
Dushyant Mehra ◽  
Santosh Adhikari ◽  
Chiranjib Banerjee ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Living Cells ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Acquisition Time ◽  
Structure And Dynamics ◽  
Chromatin Structure And Dynamics

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

scholarly journals Actin cytoskeletal inhibitor 19,20-epoxycytochalasin Q sensitizes yeast cells lacking ERG6 through actin-targeting and secondarily through disruption of lipid homeostasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kwanrutai Watchaputi ◽  
Pichayada Somboon ◽  
Nipatthra Phromma-in ◽  
Khanok Ratanakhanokchai ◽  
Nitnipa Soontorngun
Keyword(s):  
Lipid Droplets ◽  
Wild Type Strain ◽  
Yeast Cells ◽  
Lipid Homeostasis ◽  
Ergosterol Biosynthesis ◽  
Valuable Insight ◽  
Wild Type ◽  
Antifungal Property ◽  
Primary Target ◽  
Very High

AbstractRepetitive uses of antifungals result in a worldwide crisis of drug resistance; therefore, natural fungicides with minimal side-effects are currently sought after. This study aimed to investigate antifungal property of 19, 20-epoxycytochalasin Q (ECQ), derived from medicinal mushroom Xylaria sp. BCC 1067 of tropical forests. In a model yeast Saccharomyces cerevisiae, ECQ is more toxic in the erg6∆ strain, which has previously been shown to allow higher uptake of many hydrophilic toxins. We selected one pathway to study the effects of ECQ at very high levels on transcription: the ergosterol biosynthesis pathway, which is unlikely to be the primary target of ECQ. Ergosterol serves many functions that cholesterol does in human cells. ECQ’s transcriptional effects were correlated with altered sterol and triacylglycerol levels. In the ECQ-treated Δerg6 strain, which presumably takes up far more ECQ than the wild-type strain, there was cell rupture. Increased actin aggregation and lipid droplets assembly were also found in the erg6∆ mutant. Thereby, ECQ is suggested to sensitize yeast cells lacking ERG6 through actin-targeting and consequently but not primarily led to disruption of lipid homeostasis. Investigation of cytochalasins may provide valuable insight with potential biopharmaceutical applications in treatments of fungal infection, cancer or metabolic disorder.

scholarly journals Improved localization precision via restricting confined biomolecule stochastic motion in single-molecule localization microscopy

Nanophotonics ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jielei Ni ◽  
Bo Cao ◽  
Gang Niu ◽  
Danni Chen ◽  
Guotao Liang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Direct Evidence ◽  
Diffraction Limit ◽  
Drift Correction ◽  
Stochastic Motion ◽  
Localization Microscopy ◽  
Alexa Fluor ◽  
Biological Studies ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract Single-molecule localization microscopy (SMLM) plays an irreplaceable role in biological studies, in which nanometer-sized biomolecules are hardly to be resolved due to diffraction limit unless being stochastically activated and accurately located by SMLM. For biological samples preimmobilized for SMLM, most biomolecules are cross-linked and constrained at their immobilizing sites but still expected to undergo confined stochastic motion in regard to their nanometer sizes. However, few lines of direct evidence have been reported about the detectability and influence of confined biomolecule stochastic motion on localization precision in SMLM. Here, we access the potential stochastic motion for each immobilized single biomolecule by calculating the displacements between any two of its localizations at different frames during sequential imaging of Alexa Fluor-647-conjugated oligonucleotides. For most molecules, localization displacements are remarkably larger at random frame intervals than at shortest intervals even after sample drift correction, increase with interval times and then saturate, showing that biomolecule stochastic motion is detected and confined around the immobilizing sizes in SMLM. Moreover, localization precision is inversely proportional to confined biomolecule stochastic motion, whereas it can be deteriorated or improved by enlarging the biomolecules or adding a post-crosslinking step, respectively. Consistently, post-crosslinking of cell samples sparsely stained for tubulin proteins results in a better localization precision. Overall, this study reveals that confined stochastic motion of immobilized biomolecules worsens localization precision in SMLM, and improved localization precision can be achieved via restricting such a motion.

scholarly journals Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

2019 ◽  
Author(s):  
Lekha Patel ◽  
Dylan M. Owen ◽  
Edward A.K. Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Diffraction Limit ◽  
Training Data ◽  
Switching Behavior ◽  
Cellular Structures ◽  
Exact Probability ◽  
Localization Microscopy ◽  
Exact Probability Distribution ◽  
Single Molecule Localization Microscopy

AbstractMany recent advancements in single molecule localization microscopy exploit the stochastic photo-switching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging. Modeling the photo-switching behavior of a fluorophore as a continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photo-switching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a dSTORM experiment involving an arbitrary number of molecules. We demonstrate that when training data is available to estimate photo-switching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations.

scholarly journals Imaging unlabeled proteins on DNA with super-resolution

2020 ◽  
Vol 48 (6) ◽  
pp. e34-e34 ◽  
Author(s):  
Anna E C Meijering ◽  
Andreas S Biebricher ◽  
Gerrit Sitters ◽  
Ineke Brouwer ◽  
Erwin J G Peterman ◽  
...  
Keyword(s):  
Detection Limit ◽  
Single Molecule ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Current Image ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Biomolecular Analysis ◽  
Current Detection ◽  
Inverse Imaging

Abstract Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

scholarly journals A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast

2014 ◽  
Vol 206 (3) ◽  
pp. 357-366 ◽  
Author(s):  
Chao-Wen Wang ◽  
Yu-Hsuan Miao ◽  
Yi-Shun Chang
Keyword(s):  
Endoplasmic Reticulum ◽  
Stationary Phase ◽  
Lipid Droplets ◽  
Physiological State ◽  
Lipid Homeostasis ◽  
Cellular Lipid ◽  
Sterol Esters ◽  
Selective Autophagy ◽  
The Core ◽  
Atg Proteins

Stationary phase (stat-phase) is a poorly understood physiological state under which cells arrest proliferation and acquire resistance to multiple stresses. Lipid droplets (LDs), organelles specialized for cellular lipid homeostasis, increase in size and number at the onset of stat-phase. However, little is known about the dynamics of LDs under this condition. In this paper, we reveal the passage of LDs from perinuclear endoplasmic reticulum association to entry into vacuoles during the transition to stat-phase. We show that the process requires the core autophagy machinery and a subset of autophagy-related (Atg) proteins involved in selective autophagy. Notably, the process that we term stat-phase lipophagy is mediated through a sterol-enriched vacuolar microdomain whose formation and integrity directly affect LD translocation. Intriguingly, cells defective in stat-phase lipophagy showed disrupted vacuolar microdomains, implying that LD contents, likely sterol esters, contribute to the maintenance of vacuolar microdomains. Together, we propose a feed-forward loop in which lipophagy stimulates vacuolar microdomain formation, which in turn promotes lipophagy during stat-phase.

scholarly journals Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

2016 ◽  
Vol 212 (6) ◽  
pp. 621-631 ◽  
Author(s):  
Ariadna P. Velázquez ◽  
Takashi Tatsuta ◽  
Ruben Ghillebert ◽  
Ingmar Drescher ◽  
Martin Graef
Keyword(s):  
Cell Survival ◽  
Lipid Droplets ◽  
Metabolic Diseases ◽  
De Novo ◽  
Phospholipid Composition ◽  
Yeast Cells ◽  
Lipid Homeostasis ◽  
Er Homeostasis ◽  
Insight Into ◽  
Catabolic Process

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.

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