Lena K. Schroeder

Ultra-High Resolution 3D Imaging of Whole Cells

Summary Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Quantifying and Optimizing Single-Molecule Switching Nanoscopy at High Speeds

Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5–25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

Scyl1 scaffolds class II Arfs to selective subcomplexes of coatomer via the γ-COP appendage domain

ABSTRACT Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO− motif to interact directly with the appendage domain of coatomer subunit &ggr;-2 (also known as COPG2 or &ggr;2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to &ggr;2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

Scyl1 scaffolds class II Arfs to specific subcomplexes of coatomer through the γ-COP appendage domain

ABSTRACT Coatomer (COPI)-coated vesicles mediate membrane trafficking in the early secretory pathway. There are at least three subclasses of COPI coats and two classes of Arf GTPases that couple COPI coat proteins to membranes. Whether mechanisms exist to link specific Arfs to specific COPI subcomplexes is unknown. We now demonstrate that Scy1-like protein 1 (Scyl1), a member of the Scy1-like family of catalytically inactive protein kinases, oligomerizes through centrally located HEAT repeats and uses a C-terminal RKXX-COO− motif to interact directly with the appendage domain of coatomer subunit &ggr;-2 (also known as COPG2 or &ggr;2-COP). Through a distinct site, Scyl1 interacts selectively with class II Arfs, notably Arf4, thus linking class II Arfs to &ggr;2-bearing COPI subcomplexes. Therefore, Scyl1 functions as a scaffold for key components of COPI coats, and disruption of the scaffolding function of Scyl1 causes tubulation of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-Golgi, similar to that observed following the loss of Arf and Arf-guanine-nucleotide-exchange factor (GEF) function. Our data reveal that Scyl1 is a key organizer of a subset of the COPI machinery.

A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi

Besides its well-established role in generating COPI vesicles by recruiting coatomer at the Golgi, the small GTPase ARF1 is additionally involved in the formation of anterograde and retrograde tubular carriers at the Golgi.

Apical Localization of Inositol 1,4,5-Trisphosphate Receptors Is Independent of Extended Synaptotagmins in Hepatocytes

Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.

Simultaneously Measuring Image Features and Resolution in Live-Cell STED Images

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique called nested-loop ensemble PSF fitting. This approach enables extraction of the size of cellular features and the PSF width in fixed-cell and live-cell images without relying on beads or precalibration. Nested-loop ensemble PSF fitting accurately recapitulates microtubule diameter from stimulated emission depletion images and can measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. Our algorithm has been implemented as a plugin for the PYthon Microscopy Environment, a freely available and open-source software.

Nano-scale size holes in ER sheets provide an alternative to tubules for highly-curved membranes

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Super-resolution microscopy recently revealed densely packed, rapidly moving ER tubules, highlighting the importance of revisiting classical views of ER structure with high spatial resolution in living cells. Using live-cell Stimulated Emission Depletion (STED) microscopy, we show highly dynamic, subdiffraction-sized holes in ER sheets. Holes coexist with uniform sheet regions and are distinct from tubular ER structures. The curvature-stabilizing reticulon protein Rtn4 localizes to these holes and the ER luminal tether Climp63 controls their diameter and mobility. Analytical modeling demonstrates that holes in ER sheets can serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction, thus providing an explanation for how the ER locally alters its morphology on fast time-scales. One Sentence Summary Dynamic nano-scale sized holes are prominent features of ER sheets that serve as reservoirs for curvature-stabilizing proteins to support ER tubule extension and retraction.

Stimulated Emission Depletion (STED) Imaging of Clathrin-Mediated Endocytosis in Living Cells

The recent development of probes and labeling strategies for multicolor super-resolution imaging in living cells allows cell biologists to follow cellular processes with unprecedented details. Here we describe how to image endocytic events at the plasma membrane of living cells using commercial (Leica, Abberior Instruments) or custom built STED microscopes.

Land-locked mammalian Golgi reveals cargo transport between stable cisternae

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10–20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue—even when individual Golgi cisternae are separated and “land-locked” between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells.The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model.