Photoactivated Localization Microscopy Research Articles

Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs. These differences in Gag multimerization were further shown to affect downstream selective protein sorting into HIV membranes, indicating that the choice of RNA for packaging can modulate viral membrane compositions. These findings should advance the understanding of HIV assembly and further benefit the development of virus-like particle-based therapeutics.

Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy.

Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2 . Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Channelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy

AbstractMicrobial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin‐2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield. We demonstrate the SExY method on several exemplary molecular targets and explicitly introduce low-abundant protein targets (e.g. nuclear proteins such as cbp1 and mis16, and the centromere-specific histone protein cnp1). The SExY protocol optimizations increasing protein yield could be beneficial for many studies, when targeting low abundance proteins, or for studies that rely on genetic labelling for various reasons (e.g. for proteins that cannot be easily targeted by extrinsic staining or in case artefacts introduced by unspecific staining interfere with data quality).

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization.

Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties.

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62, altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

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.

Seeing Beyond the Limits With Super-Resolution Microscopy

When I was young, I wanted to be an astronaut and I was a hard-core science fiction fan. I was always drawn to heroic characters that invented something new that resulted in an amazing breakthrough. When I started my scientific career, I did not want to do something small—I wanted to do something that would be really different and impactful. This is why I chose to work on one of the most difficult problems in the field of optical (light-using) microscopy—how to see objects that are smaller than the wavelength of visible light. This meant challenging a limit that was long believed to be unbreakable. In my research, I developed a method called photoactivated localization microscopy (PALM), which enabled us to break that limit with the help of glowing (fluorescent) molecules. Using the PALM method, and other methods based on glowing molecules, scientists can learn new things about living cells and single molecules and significantly advance our understanding of life.

Photoactivatable BODIPYs for Live-Cell PALM.

Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Photoactivated localization microscopy combined with image spatial correlation analysis to study of the distribution and diffusion of Angiotensin II type 1 Receptor in the plasma membrane.

Super resolution fluorescence microscopy, (SRFM) techniques which overcome the spatial diffraction-limit has enabled us to visualize nanoscale cellular events with high temporal and spatial resolution in living cells, but in order to gain reliable mechanistic insights, a quantitative analysis is essential 1–3. In this study we used SRFM combined with novel image analysis as a powerful tool to investigate the mechanisms by which Angiotensin II type 1 Receptors (AT1Rs), are distributed and diffuse in the plasma membrane. This mechanism remains still unclear, despite its crucial role in cardiovascular homeostasis in the cell4,5. The aim of this study is to obtain quantitative information about AT1R dynamics and organization, and their corresponding receptor-ligand complexes in living cells. This was achieved by using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study nanoscale receptor distribution, a combination of expansion microscopy (ExM) and PALM was used. This study showed that AT1R lateral diffusion increased after binding to the agonist, Angiotensin II (Ang II) while no change for either the biased agonist, SII-Ang II or the antagonist, Losartan, was observed. The receptor diffusion was found to be transiently confined in the PM. In addition, ExM revealed that AT1Rs form nano clusters at the PM, and the cluster size significantly decreased after Ang II treatment. Taking these results together suggests that Ang II binding and activation cause reorganization and changes in the dynamics of AT1Rs at the PM. Our work contributes to a better comprehension of AT1R dynamic in PM and makes for a better understanding of signalling and receptor mechanisms.

Characterizing the structural rearrangement of the bacterial nucleoid by Dps.

Bacteria are subject to a wide variety of environmental stresses which requires a robust stress response system to protect the cell. A key protein in Escherichia coli known for its numerous functions in stress response and cell maintenance is the DNA-binding protein from starved cells (Dps). The Dps protein protects DNA via two different mechanisms. First, Dps oxidizes iron to prevent the generation of hydroxyl radicals. Second, in response to stress and throughout the stationary phase of the growth cycle, Dps oligomers bind to DNA and multimerize further to create a Dps-DNA complex that compacts. To access how Dps affects this structural change of DNA to afford protection of the cell's genome, photoactivatable localization microscopy is employed to generate nanometer-scale maps of the nucleoid structure throughout the E. coli growth phases. These maps are attained by super-resolving the localizations of single molecules of the nucleoid-associated protein HUα, which non-specifically binds to DNA, tagged with the photoactivatable fluorescent protein PAmCherry. Super-resolution maps of the nucleoid structure are compared to maps of the nucleoid in E. coli strains with dps deleted. Tagging Dps and other major nucleoid associated proteins with PAmCherry provides the distribution of these proteins across the nucleoid to gain insight on how and if Dps interacts with other nucleoid associated proteins. By observing these structural changes over the growth period of E. coli, insight can be gained about how changes in DNA structure can lead to protection via Dps.

Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Super-resolving microscopy reveals the localizations and movement dynamics of stressosome proteins in Listeria monocytogenes

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy.

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells, and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted tetrazine ligation and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of an imaging channel with another large Stokes shift dye have been demonstrated.

Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

A fundamental understanding of gene regulation requires a quantitative characterization of the spatial organization and dynamics of chromatin. The advent of fluorescence super-resolution microscopy techniques such as photoactivated localization microscopy (PALM) presented a breakthrough to visualize structural features with a resolution of ~20 nm in fixed cells. However, until recently the long acquisition time of super-resolution images prevented high-resolution measurements in living cells due to spreading of localizations caused by chromatin motion. Here, we present a step-by step protocol for our recently developed approach for correlatively imaging telomeres with conventional fluorescence and PALM, in order to obtain time-averaged super-resolution images and dynamic parameters in living cells. First, individual single molecule localizations are assigned to a locus as it moves, allowing to discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. By subtracting the telomere trajectory from the localization of bound molecules, the motion blurring is then corrected, and high-resolution structural characterizations can be made. These structural parameters can also be related to local chromatin motion or larger scale domain movement. This protocol therefore improves the ability to analyze the mobility and time-averaged nanoscopic structure of locus-specific chromatin with single-molecule sensitivity.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Correction Notice: Correlative Conventional and Super-resolution Photoactivated Localization Microscopy (PALM) Imaging to Characterize Chromatin Structure and Dynamics in Live Mammalian Cells.

Unveiling the Nanoscale Dynamics of the Exocytic Machinery in Chromaffin Cells with Single-Molecule Imaging.

Neuronal and hormonal communication relies on the exocytic fusion of vesicles containing neurotransmitters and hormones with the plasma membrane. This process is tightly regulated by key protein-protein and protein-lipid interactions and culminates in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation and zippering that promotes vesicular fusion. Located on both sides of the vesicle and the plasma membrane, the zippering of the SNARE complex acts to overcome the energy barrier afforded by the repulsive electrostatic force stemming from apposing two negatively charged phospholipid membranes. Another component opposing the timely organization of the fusion machinery is thermal Brownian energy that tends to homogenize all cellular molecules by constantly switching their motions and directions through short-lived molecular interactions. Much less is known of the mechanisms counteracting these chaotic forces, allowing seamless cellular functions such as exocytic fusion. Super-resolution microscopy techniques such as single-molecule imaging have proven useful to start uncovering these nanoscale mechanisms. Here, we used single-particle tracking photoactivatable localization microscopy (sptPALM) to track syntaxin-1-mEos, a SNARE protein located on the plasma membrane of cultured bovine chromaffin cells. We demonstrate that syntaxin-1-mEos undergoes dramatic change in its mobility in response to secretagogue stimulation leading to increased nanoclustering. These nanoclusters are transient in nature and likely to provide docked vesicles with a molecular environment conducive to exocytic fusion.

Angiotensin II Treatment Induces Reorganization and Changes in the Lateral Dynamics of Angiotensin II Type 1 Receptor in the Plasma Membrane Elucidated by Photoactivated Localization Microscopy Combined with Image Spatial Correlation Analysis.

The mechanisms by which angiotensin II type 1 receptor is distributed and the diffusional pattern in the plasma membrane (PM) remain unclear, despite their crucial role in cardiovascular homeostasis. In this work, we obtained quantitative information of angiotensin II type 1 receptor (AT1R) lateral dynamics as well as changes in the diffusion properties after stimulation with ligands in living cells using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study the organization of the receptor at the nanoscale, expansion microscopy (ExM) combined with PALM was performed. This study revealed that AT1R lateral diffusion increased after binding to angiotensin II (Ang II) and the receptor diffusion was transiently confined in the PM. In addition, ExM revealed that AT1R formed nanoclusters at the PM and the cluster size significantly decreased after Ang II treatment. Taking these results together suggest that Ang II binding and activation cause reorganization and changes in the dynamics of AT1R at the PM.