Clathrin Dynamics Research Articles

Stochastic Nature of Clathrin-Coated Pit Assembly

The dynamics of clathrin coated pit (CCP) formation, observed through total internal reflection fluorescence microscopy, shows considerable diversity. Foremost is fate divergence, which leads to “abortive” and “productive” pits, i.e., structures which, respectively, do or do not mature into clathrin coated vesicles (CCVs). Also, there is notable heterogeneity in the lifetimes of abortive pits and the apparent time to the completion of productive CCPs. We explore the extent to which the stochastic nature of CCP growth can explain these observations. For this purpose we analyze a simple model that includes a kinetic scheme for CCP assembly and a related functional form for free energy of CCP formation. Using this model, we calculate the lifetime distribution of abortive pits (via Monte Carlo simulation) and fit it to experimental data to determine the exact effective potential experienced by CCPs. We show that the CCPs without cargo are energetically unstable, and that the binding of cargo might stabilize a CCP and thereby facilitate CCV formation. Finally, we estimate how variation in the time of CCV formation is linked to the stochastic associations and dissociations of coat components.

Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.

Imaging Single Endocytic Events Reveals Diversity in Clathrin, Dynamin and Vesicle Dynamics

The dynamics of clathrin-mediated endocytosis can be assayed using fluorescently tagged proteins and total internal reflection fluorescence microscopy. Many of these proteins, including clathrin and dynamin, are soluble and changes in fluorescence intensity can be attributed either to membrane/vesicle movement or to changes in the numbers of individual molecules. It is important for assays to discriminate between physical membrane events and the dynamics of molecules. Two physical events in endocytosis were investigated: vesicle scission from the plasma membrane and vesicle internalization. Single vesicle analysis allowed the characterization of dynamin and clathrin dynamics relative to scission and internalization. We show that vesicles remain proximal to the plasma membrane for variable amounts of time following scission, and that uncoating of clathrin can occur before or after vesicle internalization. The dynamics of dynamin also vary with respect to scission. Results from assays based on physical events suggest that disappearance of fluorescence from the evanescent field should be re-evaluated as an assay for endocytosis. These results illustrate the heterogeneity of behaviors of endocytic vesicles and the importance of establishing suitable evaluation criteria for biophysical processes.

Real-Time Imaging of Clathrin Dynamics in Three Dimensions

Clathrin mediated endocytosis is a crucial factor in maintaining cellular dynamics. So far, real-time observation of this phenomenon has been achieved using methods that are limited to two dimensional spatial analysis even though cells have three dimensional geometries. Here we have used a fast, confocal based, three dimensional automated tracking scheme to analyze clathrin coat dynamics at the ventral and dorsal surfaces of mammalian cells. This technique presents a direct observation of a clathrin mediated virus entry at the apical surface of polarized cells for the first time.

Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits

Recently, it has become clear that the actin cytoskeleton is involved in clathrin-mediated endocytosis. During clathrin-mediated endocytosis, clathrin triskelions and adaptor proteins assemble into lattices, forming clathrin-coated pits. These coated pits invaginate and detach from the membrane, a process that requires dynamic actin polymerization. We found an unexpected role for the clathrin adaptor epsin in regulating actin dynamics during this late stage of coated vesicle formation. In Dictyostelium cells, epsin is required for both the membrane recruitment and phosphorylation of the actin- and clathrin-binding protein Hip1r. Epsin-null and Hip1r-null cells exhibit deficiencies in the timing and organization of actin filaments at clathrin-coated pits. Consequently, clathrin structures persist on the membranes of epsin and Hip1r mutants and the internalization of clathrin structures is delayed. We conclude that epsin works with Hip1r to regulate actin dynamics by controlling the spatial and temporal coupling of actin filaments to clathrin-coated pits. Specific residues in the ENTH domain of epsin that are required for the membrane recruitment and phosphorylation of Hip1r are also required for normal actin and clathrin dynamics at the plasma membrane. We propose that epsin promotes the membrane recruitment and phosphorylation of Hip1r, which in turn regulates actin polymerization at clathrin-coated pits.

A Comparison of GFP-Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)-tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP-tagged LC variants. Cy3-labeled light chains (Cy3-LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1-2 min the Cy3-LC heavy chain complexes entered clathrin-coated structures, whereas uncomplexed Cy3-LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time-course of an endocytic cycle. To explore whether GFP-tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild-type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3- and Alexa 488-labeled LC behaved similar to wild-type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.

Swa2p-dependent clathrin dynamics is critical for Flo11p processing and ‘Mat’ formation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to form complex multicellular structures called mats on low-density agar Petri plates. Mat formation strictly depends on Flo11p, a cell surface mannoprotein that mediates the adhesion of yeast cells to the agar surface. Here, we show that Swa2p, an auxilin ortholog required for clathrin-coated vesicle uncoating, is strictly required for biofilm formation. We show that the maturation and cellular levels of Flo11p are affected in Δswa2 cells, yet without compromising invasive growth. Both the TPR and J-domains of Swa2p, but not its clathrin-binding and ubiquitin-association motifs, are required for its function in Flo11p processing.

EGFR as a Tool for Probing Productive Endocytosis Events with Epi/TIRF

Following activation, many receptors at the cell surface partition into clathrin coated pits (CCPs) which subsequently pinch off and move into the cell. Total internal reflection fluorescence (TIRF) microscopy has been used to study such events because it predominantly illuminates <70nm proximal to the coverslip, reducing background fluorescence from the depth of the cell. Loss of clathrin fluorescence from the evanescent field is typically interpreted as endocytosis; however alternative clathrin dynamics at the cell membrane, including formation of clathrin coated pits (CCPs) that are later aborted, has been described. Disappearance of clathrin in TIRF could represent movement of a CCP away from the membrane, uncoating of clathrin from a CCP, or dissociation of clathrin from the cell membrane. To differentiate between these possibilities, we simultaneously imaged a marker which remains with the vesicle. The epidermal growth factor receptor (EGFR) is activated by EGF binding, which induces uptake of its receptor into the cell. In Cos7 cells we analyzed EGFR containing CCPs using Epi/TIR imaging to visualize vesicles as they moved from the cell membrane to deeper in the cell (appearing only in Epi). This allowed us to identify the CCPs that were unambiguously undergoing endocytosis. We demonstrate the utility of EGFR as a model cargo for tracking endocytic vesicles. We tracked individual vesicles following endocytosis, and examined the dynamics of clathrin and EGFR relative to each other. This reveals a range of vesicle behavior as they enter the cell including variable dynamics of clathrin uncoating.

Variability of Dynamin and Clathrin Dynamics in Clathrin Mediated Endocytosis

Clathrin mediated endocytosis (CME) is a pathway which internalizes receptors from the cell surface. The scission of clathrin coated vesicles from the plasma membrane requires dynamin. However, there are multiple models of dynamin mechanism without a consensus on the exact nature of its role. Total internal reflection fluorescence (TIRF) microscopy allows the visualization of fluorescently tagged proteins during individual endocytic events. TIRF provides better sensitivity than other techniques, however the analysis of this data remains challenging due to several factors including a low signal-to-noise ratio and an abundance of clathrin on the membrane. To overcome this problem, it is common to impose rules on the data and group intensity traces from individual clathrin spots, aligning them to a common event. We have examined the basis of these criteria, and in the experiments presented here we employed a very broad selection criteria. Using TIRF we imaged dynamin and clathrin in Cos7 cells, and characterized many individual vesicles. We observe a variety of dynamic behaviors at the cell membrane, including major differences in the time of loss of clathrin and dynamin fluorescence in individual traces. We found that grouping and aligning to a common event masked relevant differences and dynamics of the molecules with respect to each other. The time differences between clathrin and dynamin leaving the plasma membrane are not tightly correlated; these different behaviors could represent different sub-populations of membrane events, or heterogeneity within a single class of event. Our data indicates that another marker for endocytic events must be used. This will be especially important in the search for the mechanism of dynamin, to ensure that conclusions drawn from in vivo imaging studies pertain to a genuine biological action.

Analysis of Clathrin Self-Assembly by Infrared Spectroscopy

The clathrin protein self-assembles into a lattice that coats intracellular vesicles involved in sorting and transport of membrane-associated proteins. Inside a cell, clathrin self-assembly is initiated by interaction with adaptor proteins, but the basic self-assembly reaction can be recapitulated in vitro with recombinant fragments of clathrin that represent the C-terminal third of the clathrin heavy chain. This self-assembling fragment is the central portion or Hub of the three-legged clathrin triskelion. Previous studies have suggested that assembly interactions between Hub legs display micromolar affinity and likely involve hydrophobic interactions, though assembly is modulated by a pH-sensitive salt bridge. The studies to be reported investigated whether mid-infrared spectroscopy of Hubs in solutions and also in a controlled humidity environment can be used to establish additional features of Hub self-assembly and potentially as a dynamic monitor of clathrin assembly. Comparison of spectra generated from assembled and disassembled clathrin revealed that hydration plays a role in assembly and that several absorption bands (1117 and 1220 wavenumbers) were present in assembled hubs that were absent in unassembled hubs. Such spectra were obtained on a Bruker 66v/S. Spectra generated during assembly suggested that a decrease in random coil and an increase in alpha helical content occur during Hub assembly, indicative of increased thermodynamic stability achieved during lattice formation. (Preliminary Raman data was also obtained from assembled Hubs.) These results demonstrate that analysis of Hub behavior in the infrared can be informative about the dynamics of clathrin self-assembly and suggest infrared spectroscopy as a novel approach to understanding the molecular details of clathrin-coated vesicle formation.

Regulation of endosomal clathrin and retromer-mediated endosome to Golgi retrograde transport by the J-domain protein RME-8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG-14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome-associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J-domain protein RME-8 associates with the retromer component SNX-1. Loss of SNX-1, RME-8, or the clathrin chaperone Hsc70/HSP-1 leads to over-accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG-14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME-8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.

Distinct Dynamics Of Endocytic Clathrin Coated Pits And Coated Plaques

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as ‘‘coated pits’’ and ‘‘coated plaques.’’ Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane— classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular structures.

Dynamics of Arabidopsis Dynamin-Related Protein 1C and a Clathrin Light Chain at the Plasma Membrane

Plant morphogenesis depends on polarized exocytic and endocytic membrane trafficking. Members of the Arabidopsis thaliana dynamin-related protein 1 (DRP1) subfamily are required for polarized cell expansion and cytokinesis. Using a combination of live-cell imaging techniques, we show that a functional DRP1C green fluorescent fusion protein (DRP1C-GFP) was localized at the division plane in dividing cells and to the plasma membrane in expanding interphase cells. In both tip growing root hairs and diffuse-polar expanding epidermal cells, DRP1C-GFP organized into dynamic foci at the cell cortex, which colocalized with a clathrin light chain fluorescent fusion protein (CLC-FFP), suggesting that DRP1C may participate in clathrin-mediated membrane dynamics. DRP1C-GFP and CLC-GFP foci dynamics are dependent on cytoskeleton organization, cytoplasmic streaming, and functional clathrin-mediated endocytic traffic. Our studies provide insight into DRP1 and clathrin dynamics in the plant cell cortex and indicate that the clathrin endocytic machinery in plants has both similarities and striking differences to that in mammalian cells and yeast.

Auxilin is essential for Delta signaling

Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin, an endocytic factor first identified in vertebrate cells. Here, we show in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. We find that like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. We discuss these observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling.

2S2-4 Mapping the molecular dynamics of clathrin mediated endocytosis(2S2 Interactions between the cell membrane and the actin cytoskeleton: Biophysical approaches,The 46th Annual Meeting of the Biophysical Society of Japan)

2S2-4 Mapping the molecular dynamics of clathrin mediated endocytosis(2S2 Interactions between the cell membrane and the actin cytoskeleton: Biophysical approaches,The 46th Annual Meeting of the Biophysical Society of Japan)

Dynamics of clathrin and adaptor proteins during endocytosis

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for > or =30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis.

Imaging Clathrin Dynamics in Drosophila melanogaster Hemocytes Reveals a Role for Actin in Vesicle Fission

Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.

Structure and dynamics of clathrin coated vesicles

Clathrin coated vesicles are the principal route by which ligands such as transferrin and LDL as well as many viruses enter cells. The structure of a clathrin coat, derived by combining atomic structures from x-ray crystallography with moderate resolution structures from cryoEM, identifies the molecular interactions critical for coat formation. The dynamics of assembly and uncoating, studied in living cells by fluorescence microscopy, can be analyzed in mechanistic terms, with reference to the structure of the clathrin lattice and to the biochemistry of its many regulatory components.

Individual Phosphoinositide 3-Kinase C2α Domain Activities Independently Regulate Clathrin Function

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a member of the class II PI-3 kinases, defined by the presence of a second C2 domain at their C termini. The cellular functions of the class II enzymes are incompletely understood, though they have been implicated in receptor activation pathways initiated by epidermal growth factor, insulin, and chemokines. PI3K-C2alpha was recently found to be localized to clathrin-coated membranes in the trans-Golgi network and at endocytic sites on the plasma membrane. Further, a specific binding site was identified for clathrin on the N terminus of PI3K-C2alpha, whose occupancy resulted in lipid kinase activation. Expression of PI3K-C2alpha in cells dramatically affected clathrin distribution and function in cells, leading to accumulation of intracellular clathrin-coated structures, which are visualized here at the ultrastructural level, and inhibition of clathrin-mediated transport from both the plasma membrane and the trans-Golgi network. In this study we have demonstrated that the isolated clathrin binding domain of PI3K-C2alpha can drive clathrin lattice assembly and that both it and the lipid kinase activity of the protein can independently modulate clathrin distribution and function when expressed in cells. Together, these results suggest that PI3K-C2alpha employs both protein-protein interaction and localized production of 3-phosphoinositides to affect clathrin dynamics at sites of membrane budding and targeting.

In Vivo Dynamics of Clathrin and Its Adaptor-Dependent Recruitment to the Actin-Based Endocytic Machinery in Yeast

Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast. Fluorescently tagged clathrin is found in highly mobile internal trans-Golgi/endosomal structures and in smaller cortical patches. Total internal reflection fluorescence microscopy showed that cortical patches are likely endocytic sites, as clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1. Clathrin also accumulates at the cortex with internalizing alpha factor receptor, Ste2p. Cortical clathrin localizes with epsins Ent1/2p and AP180s, and its recruitment to the surface is dependent upon these adaptors. In contrast, Sla2p, End3p, Pan1p, and a dynamic actin cytoskeleton are not required for clathrin assembly or exchange but are required for the mobility, maturation, and/or turnover of clathrin-containing endocytic structures.