MINFLUX reveals dynein stepping in live neurons.

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5'-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

Molecular mechanism of GPCR spatial organization at the plasma membrane.

G-protein-coupled receptors (GPCRs) mediate many critical physiological processes. Their spatial organization in plasma membrane (PM) domains is believed to encode signaling specificity and efficiency. However, the existence of domains and, crucially, the mechanism of formation of such putative domains remain elusive. Here, live-cell imaging (corrected for topography-induced imaging artifacts) conclusively established the existence of PM domains for GPCRs. Paradoxically, energetic coupling to extremely shallow PM curvature (<1 µm-1) emerged as the dominant, necessary and sufficient molecular mechanism of GPCR spatiotemporal organization. Experiments with different GPCRs, H-Ras, Piezo1 and epidermal growth factor receptor, suggest that the mechanism is general, yet protein specific, and can be regulated by ligands. These findings delineate a new spatiomechanical molecular mechanism that can transduce to domain-based signaling any mechanical or chemical stimulus that affects the morphology of the PM and suggest innovative therapeutic strategies targeting cellular shape.

Optimal precision and accuracy in 4Pi-STORM using dynamic spline PSF models.

Coherent fluorescence imaging with two objective lenses (4Pi detection) enables single-molecule localization microscopy with sub-10 nm spatial resolution in three dimensions. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation and the challenging nature of the data analysis. Here we report the development of a 4Pi-STORM microscope, which obtains optimal resolution and accuracy by modeling the 4Pi point spread function (PSF) dynamically while also using a simpler optical design. Dynamic spline PSF models incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal evolution of the optical system. Our method reaches the theoretical limits for precision and minimizes phase-wrapping artifacts by making full use of the information content of the data. 4Pi-STORM achieves a near-isotropic three-dimensional localization precision of 2-3 nm, and we demonstrate its capabilities by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy.

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or a spontaneously blinking dye for single-molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

Photoactivatable Fluorescent Dyes with Hydrophilic Caging Groups and Their Use in Multicolor Nanoscopy.

We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity (visible or infrared) light of about twice the wavelength. The caged fluorescent dyes and labels derived therefrom display high water solubility and convert upon photoactivation into validated super-resolution and live-cell-compatible fluorophores. In combination with popular fluorescent markers, multiple (up to six)-color images can be obtained with stimulated emission depletion nanoscopy. Moreover, individual fluorophores can be localized with precision <3 nm (standard deviation) using MINSTED and MINFLUX techniques.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio. Furthermore, imaging with longer wavelengths holds promise for reducing photostress. Here, we demonstrate that a strategy based on enzymatic self-labeling of the HaloTag fusion protein by high-performance synthetic fluorophore labels provides a robust avenue to superior in vivo analysis with STED nanoscopy in the far-red spectral range. We illustrate our approach by mapping the nanoscale distributions of the abundant scaffolding protein PSD95 at the postsynaptic membrane of excitatory synapses in living mice. With silicon-rhodamine as the reporter fluorophore, we present imaging with high contrast and low background down to ∼70-nm lateral resolution in the visual cortex at ≤25-µm depth. This approach allowed us to identify and characterize the diversity of PSD95 scaffolds in vivo. Besides small round/ovoid shapes, a substantial fraction of scaffolds exhibited a much more complex spatial organization. This highly inhomogeneous, spatially extended PSD95 distribution within the disk-like postsynaptic density, featuring intricate perforations, has not been highlighted in cell- or tissue-culture experiments. Importantly, covisualization of the corresponding spine morphologies enabled us to contextualize the diverse PSD95 patterns within synapses of different orientations and sizes.

Efflux pump insensitive rhodamine-jasplakinolide conjugates for G- and F-actin imaging in living cells.

The actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes, such as SiR-actin, suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We analyzed a series of new probes composed of jasplakinolide and modified rhodamine fluorophores and found that rhodamine positional isomerism has a profound effect on probe performance. The probes based on the 6'-carboxy-carbopyronine scaffold are considerably less susceptible to efflux and allow efficient staining without efflux pump inhibitors. They can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We show that jasplakinolide-based fluorescent probes bind not only to actin filaments, but also to G-actin, which enables imaging highly dynamic actin structures. We demonstrate an excellent performance of the new probes in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

Ultrastructural anatomy of nodes of Ranvier in the peripheral nervous system as revealed by STED microscopy.

We used stimulated emission depletion (STED) superresolution microscopy to analyze the nanoscale organization of 12 glial and axonal proteins at the nodes of Ranvier of teased sciatic nerve fibers. Cytoskeletal proteins of the axon (betaIV spectrin, ankyrin G) exhibit a high degree of one-dimensional longitudinal order at nodal gaps. In contrast, axonal and glial nodal adhesion molecules [neurofascin-186, neuron glial-related cell adhesion molecule (NrCAM)] can arrange in a more complex, 2D hexagonal-like lattice but still feature a ∼190-nm periodicity. Such a lattice-like organization is also found for glial actin. Sodium and potassium channels exhibit a one-dimensional periodicity, with the Nav channels appearing to have a lower degree of organization. At paranodes, both axonal proteins (betaII spectrin, Caspr) and glial proteins (neurofascin-155, ankyrin B) form periodic quasi-one-dimensional arrangements, with a high degree of interdependence between the position of the axonal and the glial proteins. The results indicate the presence of mechanisms that finely align the cytoskeleton of the axon with the one of the Schwann cells, both at paranodal junctions (with myelin loops) and at nodal gaps (with microvilli). Taken together, our observations reveal the importance of the lateral organization of proteins at the nodes of Ranvier and pave the way for deeper investigations of the molecular ultrastructural mechanisms involved in action potential propagation, the formation of the nodes, axon-glia interactions, and demyelination diseases.

Adaptive-illumination STED nanoscopy.

The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to ∼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.

Characterization of prion protein function by focal neurite stimulation.

The cellular prion protein (PrPC), encoded by the PRNP gene, is a ubiquitous glycoprotein, which is highly expressed in the brain. This protein, mainly known for its role in neurodegenerative diseases, is involved in several physiological processes including neurite outgrowth. By using a novel focal stimulation technique, we explored the potential function of PrPC, in its soluble form, as a signaling molecule. Thus, soluble recombinant prion proteins (recPrP) encapsulated in micro-vesicles were released by photolysis near the hippocampal growth cones. Local stimulation of wild-type growth cones with full-length recPrP induced neurite outgrowth and rapid growth cone turning towards the source. This effect was shown to be concentration dependent. Notably, PrPC-knockout growth cones were insensitive to recPrP stimulation, but this property was rescued in PrP-knockout growth cones expressing GFP-PrP. Taken together, our findings indicate that recPrP functions as a signaling molecule, and that its homophilic interaction with membrane-anchored PrPC might promote neurite outgrowth and facilitate growth cone guidance.

Cell-Permeant Large Stokes Shift Dyes for Transfection-Free Multicolor Nanoscopy.

We designed cell-permeant red-emitting fluorescent dye labels with >140 nm Stokes shifts based on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-imino-10-germaxanthone fluorophores. The corresponding probes selectively targeting mitochondria, lysosomes, and F-actin demonstrate low toxicity and enable stimulated emission depletion (STED) nanoscopy in neurons, human fibroblasts, U2OS, and HeLa cells. In combination with known small Stokes shift dyes, our probes allow live-cell three-color STED nanoscopy of endogenous targets on popular setups with 775 nm STED wavelength.

Fluorogenic probes for live-cell imaging of the cytoskeleton.

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.

Fluorogenic Probes for Multicolor Imaging in Living Cells.

Here we present a far-red, silicon-rhodamine-based fluorophore (SiR700) for live-cell multicolor imaging. SiR700 has excitation and emission maxima at 690 and 715 nm, respectively. SiR700-based probes for F-actin, microtubules, lysosomes, and SNAP-tag are fluorogenic, cell-permeable, and compatible with superresolution microscopy. In conjunction with probes based on the previously introduced carboxy-SiR650, SiR700-based probes permit multicolor live-cell superresolution microscopy in the far-red, thus significantly expanding our capacity for imaging living cells.

Nanoscopy with more than 100,000 'doughnuts'.

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 µm × 100 µm-sized fields of view in <1 s using 116,000 such doughnuts.

Dual channel RESOLFT nanoscopy by using fluorescent state kinetics.

We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 µm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.