SummaryTotal 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.

No abstract

. Real-time analysis of clathrin-mediated endocytosis during cell migration. J. Cell Sci. 116, 847-855.In both the online and print versions of this paper, in Materials and Methods, the construct pEGFP-dynamin2 was incorrectly identified as a human isoform. It is a rat isoform. AUTHOR CORRECTION IntroductionThe internalization of integral membrane proteins from the cell surface through receptor-mediated endocytosis occurs via clathrin-coated pits (Schmid, 1997;Takei and Haucke, 2001). Examples of molecules that undergo clathrin-mediated endocytosis include nutrients such as iron, via transferrin uptake (Hopkins et al., 1985;Conrad et al., 1999), growth factors and cytokines, through receptors such as the epidermalgrowth-factor receptor (Lamaze and Schmid, 1995;Carter and Sorkin, 1998), and cellular adhesion molecules such as integrins (Raub and Kuentzel, 1989). Additionally, numerous pathogenic organisms and molecules have been observed to gain entry into cells by exploiting the endocytosis machinery (Doxsey et al., 1987;Sieczkarski and Whittaker, 2002). Endocytosis has also been implicated in cell migration (Bretscher, 1996;Palecek et al., 1996;Sheetz et al., 1999;Bajno et al., 2000;Kamiguchi and Lemmon, 2000). In particular, it has been proposed that an increased rate of endocytosis at the trailing edge would contribute to the polarized cycling of either bulk membrane or cellular adhesion molecules, such as integrins, to the leading edge (Bretscher, 1996;Palecek et al., 1996;Sheetz et al., 1999).Great strides have recently been made in the analysis of the different factors involved in the production of clathrin-coated vesicles (Takei and Haucke, 2001). The presence and interaction of such components as the AP-2 adapter complex (Benmerah et al., 1998;Kamiguchi et al., 1998) and the accessory proteins AP180 (Takei and Haucke, 2001), Eps15 (Benmerah et al., 1998;Benmerah et al., 1999) and Hip1R (Engqvist-Goldstein et al., 2001) are beginning to be well characterized. Many of the proteins that are involved in clathrin-mediated endocytosis have been identified and cloned, their crystal structures have been determined (ter Haar et al., 1998;Collins et al., 2002), and detailed models for the production and internalization of clathrin-coated vesicles have been suggested (Schmid, 1997;Takei and Haucke, 2001;Kirchhausen, 2002). However, numerous questions regarding the events and interactions relevant to endocytosis remain unanswered. For example, it is not clear whether activated receptors recruit AP-2 which results in the polymerization of clathrin coat components, or if clathrin-coated pits are preformed and recruit activated receptors. Additionally, the precise molecular function(s) of the GTPase dynamin in endocytosis remains to be resolved (Takei et al., 1995;Cao et al., 1998;McNiven et al., 2000;Ochoa et al., 2000;Schmid and Sorkin, 2002;Tsuboi et al., 2002). Although previous studies have focused on the neuron-specific dynamin1 isoform (Tsuboi et al., 2002), there is also a functional role in endocytosis for the...

Using total internal reflection fluorescence microscopy, we have developed an assay to monitor individual fusion events between proteoliposomes containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a supported planar bilayer containing cognate target SNAREs. Approach, docking, and fusion of individual vesicles to the target membrane were quantified by delivery and subsequent lateral spread of fluorescent phospholipids from the vesicle membrane into the target bilayer. Fusion probability was increased by raising divalent cations (Ca 2؉ and Mg 2؉ ). Fusion of individual vesicles initiated in <100 ms after the rise of Ca 2؉ and membrane mixing was complete in 300 ms. Removal of the N-terminal H abc domain of syntaxin 1A increased fusion probability >30-fold compared to the full-length protein, but even in the absence of the H abc domain, vesicle fusion was still enhanced in response to Ca 2؉ increase. Our observations establish that the SNARE core complex is sufficient to fuse two opposing membrane bilayers at a speed commensurate with most membrane fusion processes in cells. This real-time analysis of single vesicle fusion opens the door to mechanistic studies of how SNARE and accessory proteins regulate fusion processes in vivo.

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.

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.

Vimentin intermediate filaments-structures that were considered extremely stable-are in fact very dynamic. Filaments are actively transported throughout the cell along microtubules. In addition, filaments undergo constant rearrangement by robust severing and reannealing but never completely disassemble and reassemble.

SUMMARY Background Multiple intracellular transport pathways drive the formation, maintenance and function of cilia, a compartmentalised organelle associated with motility, chemo-/mechano-/photo-sensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the aetiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate if endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. Results Here we show that localisation of the clathrin light chain, AP-2 clathrin adaptor, dynamin and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function, and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. Conclusions These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume, and suggest that membrane retrieval from these compartments is counter-balanced by BBS-8 and RAB-8-mediated membrane delivery.