We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to È2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing 1, 2. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling1 -3. Focal adhesions consist of a complex network 4 of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque5 , 6 linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine7 ,8 . However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) 9 to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ~40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino-and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.Modern understanding of cellular function is founded on the revolution in the 1950s to 1970s in visualizing cellular ultrastructure by electron microscopy 10,11 . Together with the
The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.
We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.fluorescence microscopy ͉ interferometry ͉ PALM ͉ photoactivated localization microscopy ͉ single molecule imaging A fundamental question in biomedical research is how specific, nanometer-scale biomolecules are organized into multicomponent micron-scale structural and signaling ensembles that facilitate cell function. For example, microtubules are built of 8-nm tubulin subunits that incorporate on the ultrastructural level into polymers 25 nm in diameter and Ͼ10 m in length that serve as the building blocks of superstructures such as mitotic spindles and flagella. However, key challenges remain for determining cellular ultrastructure with high molecular specificity. Because cellular structures are organized on the nanoscale, nanometer resolution is required. Immunoelectron microscopy (EM)-based approaches provide the necessary resolution, but they lack robust molecular specificity because the large size of the antibodies hampers their penetration into dense structures and the specificity of the antibody can be compromised by cross-reactivity and epitope masking caused by the harsh fixation often used for high-resolution EM. Fluorescence microscopy coupled with fluorescent protein (FP) fusion technology enables imaging cellular structure with exquisite molecular specificity, but the resolution of 3D images is diffraction-limited to Ϸ200 nm in the lateral and Ϸ500 nm in the axial direction, limiting conventional fluorescence to the characterization of cellular superstructure. Some of the recent fluorescence-based superresolution microscopy techniques (1-5) demonstrated a resolution of Ͻ100 nm in the vertical direction; however, this is still insufficient to bridge the resolution gap between cellular ultrastructure and superstructure. To achieve near-ultrastructural 3D resolution even for the limited photon outputs of highmolecular-specificity FPs, we have developed a single-photon multiphase interferometric scheme and integrated it with a lateral photoactivated localization microscopy (PALM) (6
Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca 2+ dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER-plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER-PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies.FIB-SEM | thin ER | Stim1 | spine apparatus | subsurface cisternae
Highlights d Hyperactive neurons release excess FAs in lipid particles associated with ApoE d Astrocytes endocytose neuron-derived lipid particles, delivering the FAs to LDs d Astrocytes with LDs upregulate metabolic and detoxification genes d Neural activity triggers astrocytic consumption of FAs by mitochondrial oxidation
Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.DOI: http://dx.doi.org/10.7554/eLife.26975.001
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.