fabricating-optical-quality-glass-surfaces-to-study-macrophage-fusion

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

European Molecular Biology Laboratory

The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein-coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)-based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

In this chapter, the authors outline in full detail, an uncomplicated approach that enables the combination of wide-field fluorescence super-resolution microscopy with electron tomography, thereby providing an approach that affords the best possible confidence in the structures investigated. The methodical steps to obtain these high-throughput correlative nanoscopic arrays will be visually explored and outlined in detail. The authors will demonstrate the feasibility of the method on cultured Caco-2 colorectal cancer cells that are labeled for filamentous actin. The presented images, morphometric data, and generated models illustrate the strengths of our correlative approach for future advanced structural-biology-oriented questions. Correlative nanoscopy applications can be readily found in which there is a need to reveal biomolecular information at unprecedented resolution on subcellular behavior in various biological and pathobiological processes.

Combining wide-field super-resolution microscopy and electron tomography: rendering nanoscopic correlative arrays on subcellular architecture.

Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.

Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging.

Clathrin mediated endocytosis (CME) is the main route of receptor internalization in mammalian cells and this well conserved mechanism has been intensively studied for over 40yrs. In the general or 'canonical' model of CME clathrin coated pits form stochastically at the plasma membrane and coated pit curvature develops as the coated pit grows through clathrin polymerization. However, the canonical model of CME does not explain the diversity of endocytically active clathrin coated structures (CCSs) found at the plasma membrane by both electron and light microscopy. In this review we examine the canonical model of CME, highlight discrepancies with published experimental data and suggest future avenues of exploration while paying particular attention to the relationship between clathrin coated pits, plaques, sites of adhesion and the formation of endocytic 'hotspots'.

Clathrin coated pits, plaques and adhesion.

A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

Ultrastructural Characterization of Zika Virus Replication Factories.

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Article	Year
Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis. Molecular biology of the cell\| PubMed ID: 25079691	2014
Combining wide-field super-resolution microscopy and electron tomography: rendering nanoscopic correlative arrays on subcellular architecture. Methods in cell biology\| PubMed ID: 25287840	2014
Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging. Virology journal\| PubMed ID: 25428656	2014
Clathrin coated pits, plaques and adhesion. Journal of structural biology\| PubMed ID: 27431447	2016
Ultrastructural Characterization of Zika Virus Replication Factories. Cell reports\| PubMed ID: 28249158	2017
Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28842890	2017
Erratum to: Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins, the Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29151226	2017

Marko Lampe

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