Martin Schorb

Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

New methodology improves the spatial resolution and sensitivity of correlative light and EM tomography, revealing new insights into dynamic cellular processes.

Plasma Membrane Reshaping during Endocytosis Is Revealed by Time-Resolved Electron Tomography

Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are ∼100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.

Endocytic sites mature by continuous bending and remodeling of the clathrin coat

Bend me, shape me: Clathrin in action Endocytic clathrin-coated pits were among the first cellular structures described by electron microscopy over five decades ago. Despite this, the question remains: Does clathrin bind to the membrane as a flat lattice and then bend during coated pit invagination, or does clathrin assemble with a defined curvature as membranes invaginate? Avinoam et al. applied two state-of-the-art imaging approaches to resolve this conflict. They suggest that clathrin assembles into a defined flat lattice early in endocytosis, which predetermines the size of the vesicle. The assembled clathrin coat then rearranges through dynamic exchange of clathrin with the cytosolic pool to wrap around the forming vesicle. Science, this issue p. 1369 The clathrin lattice appears to be remodeled during coated vesicle budding. During clathrin-mediated endocytosis (CME), plasma membrane regions are internalized to retrieve extracellular molecules and cell surface components. Whether endocytosis occurs by direct clathrin assembly into curved lattices on the budding vesicle or by initial recruitment to flat membranes and subsequent reshaping has been controversial. To distinguish between these models, we combined fluorescence microscopy and electron tomography to locate endocytic sites and to determine their coat and membrane shapes during invagination. The curvature of the clathrin coat increased, whereas the coated surface area remained nearly constant. Furthermore, clathrin rapidly exchanged at all stages of CME. Thus, coated vesicle budding appears to involve bending of a dynamic preassembled clathrin coat.

Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity.

Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals.

The Structures of COPI-Coated Vesicles Reveal Alternate Coatomer Conformations and Interactions

COPy Coat COPI-coated vesicles are responsible for intracellular vesicular transport both within the Golgi and between the Golgi and endoplasmic reticulum. By applying subtomogram averaging from cryoelectron tomography data, Faini et al. (p. 1451, published online 24 May) were able to describe the complete three-dimensional structure for COPI-coated vesicles generated in a cell-free, membrane-budding reaction. The structures of multiple individual vesicles reveal assembly principles based on interactions that, unlike those for clathrin-coated vesicles, are not regular: The basic subunit can undergo significant conformational changes and assemble with different stoichiometries. This variability may allow the regulation of membrane curvature and vesicle size. Furthermore, forming a complete closed coat was not required to produce budded vesicles. The flexible coatomer complex makes contact with a variable number of neighbors and coats vesicles of variable size. Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.

Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers.

The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions.

Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly

Significance HIV-1 undergoes a two-step assembly process. First, an immature noninfectious particle is assembled, which leaves the infected cell. Second, the structural protein, Gag, is cleaved in the virus by the viral protease, and this leads to formation of the infectious virus. The immature virus particle therefore represents the key intermediate in HIV-1 assembly. There is currently no high-resolution information available on the arrangement of Gag within immature HIV-1. We have assembled part of HIV-1 Gag in vitro to form immature virus-like tubular protein arrays, and have solved a subnanometer-resolution structure of these arrays by using cryo-EM and tomography. This structure reveals interactions of the C-terminal capsid domain of Gag that are critical for HIV-1 assembly. The assembly of HIV-1 is mediated by oligomerization of the major structural polyprotein, Gag, into a hexameric protein lattice at the plasma membrane of the infected cell. This leads to budding and release of progeny immature virus particles. Subsequent proteolytic cleavage of Gag triggers rearrangement of the particles to form mature infectious virions. Obtaining a structural model of the assembled lattice of Gag within immature virus particles is necessary to understand the interactions that mediate assembly of HIV-1 particles in the infected cell, and to describe the substrate that is subsequently cleaved by the viral protease. An 8-Å resolution structure of an immature virus-like tubular array assembled from a Gag-derived protein of the related retrovirus Mason–Pfizer monkey virus (M-PMV) has previously been reported, and a model for the arrangement of the HIV-1 capsid (CA) domains has been generated based on homology to this structure. Here we have assembled tubular arrays of a HIV-1 Gag-derived protein with an immature-like arrangement of the C-terminal CA domains and have solved their structure by using hybrid cryo-EM and tomography analysis. The structure reveals the arrangement of the C-terminal domain of CA within an immature-like HIV-1 Gag lattice, and provides, to our knowledge, the first high-resolution view of the region immediately downstream of CA, which is essential for assembly, and is significantly different from the respective region in M-PMV. Our results reveal a hollow column of density for this region in HIV-1 that is compatible with the presence of a six-helix bundle at this position.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.Ultrastructural analysis of nuclear membrane topology and assembling NPCs reveals how mitotic cells can rapidly establish a closed nuclear compartment while at the same time making it transport competent.