ULK1 seen at the single-molecule level during autophagy initiation.

Macroautophagy/autophagy research often involves overexpressing proteins to investigate their localization, function and activity. However, this approach can disturb the inherent balance of cellular components, potentially affecting the integrity of the autophagy process. With the advent of genome-editing techniques like CRISPR-Cas9, it is now possible to tag endogenous proteins with fluorescent markers, enabling the study of their behaviors under more physiologically relevant conditions. Nevertheless, conventional microscopy methods have limitations in characterizing the behaviors of proteins expressed at endogenous levels. This challenge can be overcome by single-molecule localization microscopy (SMLM) methods, which provide single-molecule sensitivity and super-resolution imaging capabilities. In our recent study, we used SMLM in combination with genome editing to explore the behavior of endogenous ULK1 during autophagy initiation, yielding unprecedented insights into the autophagy initiation process.Abbreviation: ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; ER: endoplasmic reticulum; GABARAPL1: GABA type A receptor associated protein like 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; PALM: photo-activated localization microscopy; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P: phosphatidylinositol-3-phosphate; SMLM: single-molecule localization microscopy; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.

Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

Organelle interactions compartmentalize hepatic fatty acid trafficking and metabolism.

Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity.

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells.

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 photoactivated localization microscopy (PALM) 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 unbound dCas9 molecules, whose mobilities 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 move faster and 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 sensitivity and yields unprecedented insights across length and time scales.

Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of 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 (FA) activating enzyme Faa4 on LDs in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs colocalize with the endoplasmic reticulum (ER) where their emergence and expansion are 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 FA chain-lengths. When lipolysis is induced by refreshed medium, Faa4 shuttles through ER- and lipophagy to the vacuole, where it may activate FAs for membrane expansion and degrade Faa4 to reset its cellular abundance to levels in the log phase.

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

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.

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.

Precisely calibrated and spatially informed illumination for conventional fluorescence and improved PALM imaging applications.

Spatial light modulation using cost efficient digital micromirror devices (DMD) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, precise illumination by DMDs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMD and camera coordinate systems caused by optical components in the excitation and emission path. Here we develop a fast and easy to implement calibration procedure that determines these distortions and matches the DMD and camera coordinate system with a precision below the optical diffraction limit. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to a rigid transformation allowed by manual alignment of the DMD. We first demonstrate the application of our precisely calibrated light modulation by performing a proof of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP in budding yeast (S. cerevisiae). Next, we develop a spatially informed photoactivation approach for SMLM in which only regions of the cell that contain photoactivatable fluorescent proteins are selected for photoactivation. The reduced exposure of the cells to 405 nm light increased the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in cell morphology. As a result, the mean number of reliable single-molecule localizations was also significantly increased by 28%. Since the localization precision and the ability for single-molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatially informed photoactivation significantly improves the quality of SMLM images and single-molecule tracking data. Our precise calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.

Single-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells.

Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.

Single-Molecule Localization Microscopy with the Fluorescence-Activating and Absorption-Shifting Tag (FAST) System.

We develop and employ the Fluorescence-Activating and absorption-Shifting Tag (FAST) system for super-resolution (SR) imaging and single-molecule tracking based on single-molecule localizations. The fast off rate of fluorogen binding, combined with its spatially well-separated labeling of the densely expressed FAST fusion proteins, allowed single-molecule measurements to be performed in both living and fixed cells. The well-separated fluorescence localization density was achieved by either reversibly controlling the fluorogen concentration or by irreversibly photobleaching the FAST-fluorogen complex. The experimentally determined resolution of 28 nm allowed us to resolve Ensconsin-labeled microtubules and to track single molecules in mitochondria. Our results demonstrate that FAST is well-suited for single-molecule localization microscopy (SMLM). The small size and the availability of spectrally distinct fluorogens present unique advantages of the FAST system as a potential orthogonal labeling strategy that could be applied in conjunction with existing super-resolution dyes and photoactivatable proteins in versatile imaging applications.

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms.

Heteromeric interactions between the catalytically impaired human epidermal growth factor receptor (HER3/ERBB3) and its catalytically active homologs EGFR and HER2 are essential for their signaling. Different ligands can activate these receptor pairs but lead to divergent signaling outcomes through mechanisms that remain largely unknown. We used stochastic optical reconstruction microscopy (STORM) with pair-correlation analysis to show that EGF and neuregulin (NRG) can induce different extents of HER3 clustering that are dependent on the nature of the coexpressed HER receptor. We found that the presence of these clusters correlated with distinct patterns and mechanisms of receptor phosphorylation. NRG induction of HER3 phosphorylation depended on the formation of the asymmetric kinase dimer with EGFR in the absence of detectable higher-order oligomers. Upon EGF stimulation, HER3 paralleled previously observed EGFR behavior and formed large clusters within which HER3 was phosphorylated via a noncanonical mechanism. HER3 phosphorylation by HER2 in the presence of NRG proceeded through still another mechanism and involved the formation of clusters within which receptor phosphorylation depended on asymmetric kinase dimerization. Our results demonstrate that the higher-order organization of HER receptors is an essential feature of their ligand-induced behavior and plays an essential role in lateral cross-activation of the receptors. We also show that HER receptor ligands exert unique effects on signaling by modulating this behavior.

Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding.

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.

Super-Resolution Microscopy: From Single Molecules to Supramolecular Assemblies.

Super-resolution microscopy (SRM) methods have allowed scientists to exceed the diffraction limit of light, enabling the discovery and investigation of cellular structures at the nanometer scale, from individual proteins to entire organelles. In this review we survey the application of SRM in elucidating the structure of macromolecules in the native cellular environment. We emphasize how SRM can generate molecular maps of protein complexes and extract quantitative information on the number, size, distribution, and spatial organization of macromolecules. We discuss both the novel information that can be generated through SRM as well as the experimental considerations to examine while conducting such studies. With the increasing popularity of SRM in the biological sciences, this review will serve as a tool to navigate the range of applications and harness the power of SRM to elucidate biological structures.

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control.

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory.

Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.

Single-molecule mechanoenzymatics.

The ability of cellular signaling networks to sense, process, and respond to internal and external stimuli relies on their specific detection and transduction based on molecular recognition. The molecular mechanisms by which force is specifically sensed by mechanoenzymatic processes, translated into biochemical signals, and wired to cellular signaling networks recently became accessible with single-molecule force spectroscopy. By stretching such mechanobiochemical converters along their natural reaction coordinate, complex mechanical activation pathways and subsequent biochemical reactions may be measured in a dynamic and highly precise manner. The discovered mechanisms have in common well-tuned force-induced conformational changes that lead to exposure of active recognition sites. Newly developed strategies allow investigators to test different conformational states for activity and to elucidate mechanical architectures leading to highly specific mechanical activation pathways. Here, we discuss the advances in the new field of single-molecule mechanoenzymatics and highlight complementary examples studied in bulk and in vivo.

Functional assembly of aptamer binding sites by single-molecule cut-and-paste.

Bottom up assembly of functional molecular ensembles with novel properties emerging from composition and arrangement of its constituents is a prime goal of nanotechnology. By single-molecule cut-and-paste we assembled binding sites for malachite green in a molecule-by-molecule assembly process from the two halves of a split aptamer. We show that only a perfectly joined binding site immobilizes the fluorophore and enhances the fluorescence quantum yield by several orders of magnitude. To corroborate the robustness of this approach we produced a micrometer-sized structure consisting of more than 500 reconstituted binding sites. To the best of our knowledge, this is the first demonstration of one by one bottom up functional biomolecular assembly.

A conditional gating mechanism assures the integrity of the molecular force-sensor titin kinase.

As more and more recent investigations point out, force plays an important role in cellular regulation mechanisms. Biological responses to mechanical stress are often based on force-induced conformational changes of single molecules. The force sensor, titin kinase, is involved in a signaling complex that regulates protein turnover and transcriptional adaptation in striated muscle. The structural architecture of such a force sensor determines its response to force and must assure both activity and mechanical integrity, which are prerequisites for its function. Here, we use single-molecule force-clamp spectroscopy to show that titin kinase is organized in such a way that the regulatory domains have to unfold before secondary structure elements that determine the overall fold and catalytic function. The stepwise unfolding over many barriers with a topologically determined sequence assures that the protein can react to force by conformational changes while maintaining its structural integrity.

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding.

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM-SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.