Joshua Z. Rappoport

Imaging with total internal reflection fluorescence microscopy for the cell biologist

Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

Real-time analysis of clathrin-mediated endocytosis during cell migration.

Simultaneous dual-color total-internal-reflection fluorescence microscopy (TIR-FM) was performed to analyze the internalization and distribution of markers for clathrin-mediated endocytosis (clathrin, dynamin1, dynamin2 and transferrin) in migrating cells. In MDCK cells, which endogenously express dynamin2, the dynamin2-EGFP fluorescence demonstrated identical spatial and temporal behavior as clathrin both prior to and during internalization. By contrast, in the same cells, the neuronal dynamin1 only localized with clathrin just prior to endocytosis. In migrating cells, each endocytic marker was polarized towards the leading edge, away from the lagging edge. These observations suggest a re-evaluation of the functional differences between dynamin1 and dynamin2, and of the role of clathrin-mediated endocytosis in cell migration.

Focusing on clathrin-mediated endocytosis

Investigations into the mechanisms which regulate entry of integral membrane proteins, and associated ligands, into the cell through vesicular carriers (endocytosis) have greatly benefited from the application of live-cell imaging. Several excellent recent reviews have detailed specific aspects of endocytosis, such as entry of particular cargo, or the different routes of internalization. The aim of the present review is to highlight how advances in live-cell fluorescence microscopy have affected the study of clathrin-mediated endocytosis. The last decade has seen a tremendous increase in the development and dissemination of methods for imaging endocytosis in live cells, and this has been followed by a dramatic shift in the way this critical cellular pathway is studied and understood. The present review begins with a description of the technical advances which have permitted new types of experiment to be performed, as well as potential pitfalls of these new technologies. Subsequently, advances in the understanding of three key endocytic proteins will be addressed: clathrin, dynamin and AP-2 (adaptor protein 2). Although great strides have clearly been made in these areas in recent years, as is often the case, each answer has bred numerous questions. Furthermore, several examples are highlighted where, because of seemingly minor differences in experimental systems, what appear at first to be very similar studies have, at times, yielded vastly differing results and conclusions. Thus this is an exceedingly exciting time to study endocytosis, and this area serves as a clear demonstration of the power of applying live-cell imaging to answer fundamental biological questions.

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

Hepatitis C Virus Induces CD81 and Claudin-1 Endocytosis

ABSTRACT Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.

Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots

The removal of the epidermal growth factor receptor (EGFR) from the cell surface by endocytosis is triggered by receptor activation, but many facets of EGFR trafficking remain unresolved. We employed total internal fluorescence microscopy to elucidate the dynamics of activated EGFR at the cell surface through live-cell imaging. The results of these studies demonstrate that: (1) EGFR does not localize to caveolae in live cells either before or after activation; (2) EGFR does localize to clathrin-coated pits, but only after activation; (3) activation does not result in the formation of new clathrin-coated pits; (4) activated EGFR clusters at sites of preformed clathrin lattices; (5) The AP-2 complex is involved in the internalization of activated EGFR. Using imaging techniques to show the endocytic sorting of activated EGFR for the first time in live cells, these studies suggest a refinement of the model for EGFR entry.

Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization

Increasing production and application of nanomaterials raises significant questions regarding the potential for cellular entry and toxicity of nanoparticles. It was observed that the presence of serum reduces the cellular association of 20 nm carboxylate-modified fluorescent polystyrene beads up to 20-fold, relative to cells incubated in serum-free media. Analysis by confocal microscopy demonstrated that the presence of serum greatly reduces the cell surface association of nanoparticles, as well as the potential for internalization. However, both in the presence and absence of serum, nanoparticle entry depends upon clathrin-mediated endocytosis. Finally, experiments performed with cells cooled to 4°C suggest that a proportion of the accumulation of nanoparticles in cells was likely due to direct permeabilization of the plasma membrane.

The AP-2 Complex Is Excluded from the Dynamic Population of Plasma Membrane-associated Clathrin

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (α- and β2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.

Constitutive clathrin-mediated endocytosis of CTLA-4 persists during T cell activation

Background: CTLA-4 is an essential regulator of T cell immune responses with unusual intracellular trafficking. Results: Endocytosis of CTLA-4 is continuous with subsequent recycling and degradation. Conclusion: Clathrin-mediated endocytosis of CTLA-4 persists in activated T cells. Significance: This alters our understanding of CTLA-4 behavior and, therefore, how it might function. CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface transmembrane ligands in that CTLA-4 is rapidly internalized from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analyzed in detail the internalization, recycling, and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalized from the plasma membrane in a clathrin- and dynamin-dependent manner driven by the well characterized YVKM trafficking motif. Furthermore, we show that once internalized, CTLA-4 co-localizes with markers of recycling endosomes and is recycled to the plasma membrane. Although we observed limited co-localization of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression; however, this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalized in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane; however, CTLA-4 endocytosis continues and is not stabilized during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.

An agarose spot assay for chemotactic invasion.

The directed motility of cells toward the source of a soluble chemical (chemotaxis) plays a role in events ranging from immune function to cancer progression. Numerous chemotaxis assays are commonly employed, yet none provides an optimal combination of the relevant parameters. The ideal chemotaxis assay for use in the analysis of cells crawling across a planar surface should be cost-effective, simple to perform, and suitable for high-throughput multiplexing, as well as permit alteration of experimental conditions during cell motility. Here we describe a novel chemotaxis assay based upon the invasion of cells into agarose spots into which chemoattractants are suspended. Our studies demonstrate that this system assays chemotaxis and not chemokinesis, and provide proof-of-principle for drug screening studies as well as analysis through high-resolution cellular imaging.