Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells

Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associated genes.     

Missing-in-metastasis protein promotes internalization of magnetic nanoparticles via association with clathrin light chain and Rab7

Background

Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM.

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.     

Expression and localization of clathrin heavy chain in Drosophila melanogaster

Clathrin-coated vesicles mediate cellular endocytosis of nutrients and molecules that are involved in a variety of biological processes. Basic components of the vesicle coat are clathrin heavy chain (Chc) and clathrin light chain molecules. In Drosophila melanogaster the chc gene function has been analyzed in a number of previous studies mainly using genetic approaches. However, the chc mRNA and protein expression patterns have not been studied systematically. We have generated an antibody that specifically recognizes Chc and we have analyzed chc RNA and protein expression patterns throughout embryonic and larval stages. We found that chc mRNA and protein are highly expressed from early stages of embryogenesis onwards, consistent with genetic studies predicting a maternal contribution of the gene function. During subsequent stages mRNA and protein are co-expressed in all embryonic cells; however we found an up-regulation in specific tissues including the gut, the salivary glands, tracheal system and the epidermis. In addition the central nervous system and the nephrocyte-like garland cells show strong Chc expression at late embryogenesis. In larvae Chc is highly expressed in garland cells, imaginal discs, fat body, salivary glands and the ring gland. Subcellularly, we found Chc protein in a vesicle-like pattern within the cytoplasm and at the plasma membrane. Co-labeling studies show that Chc is partially in contact with the trans-Golgi network and co-localizes with markers for early endocytosis. Together, the antibody may serve as a new tool to study the function of Chc in clathrin-dependent cellular processes, such as endocytosis.     

Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2

The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca²⁺ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor β-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.     

A kinetic view of clathrin assembly and endocytic cargo sorting

Specificity and sensitivity in biochemical reactions can be achieved through regulation of equilibrium binding affinity or through proofreading mechanisms that allow for the dissociation of unwanted intermediates. In this essay, we aim to provide our perspectives on how the concept of kinetic proofreading might apply in the context of cargo sorting in clathrin-mediated endocytosis.     

Towards understanding the molecular mechanism of the endocytosis-like process in the bacterium Gemmata obscuriglobus

An endocytosis-like process of protein uptake in the planctomycete Gemmata obscuriglobus is a recently discovered process unprecedented in the bacterial world. The molecular mechanisms underlying this process are not yet characterized. A homolog of the MC (membrane-coating) proteins of eukaryotes has been proposed to be involved in the mechanism of this process, but its relationship to eukaryote proteins is controversial. However, a number of other proteins of G. obscuriglobus with domains homologous to those involved in endocytosis in eukaryotes can also be identified. Here we critically evaluate current bioinformatic knowledge, and suggest practical experimental steps to overcome the limits of bioinformatics in elucidating the molecular mechanism of endocytosis in bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Role of lipids and actin in the formation of clathrin-coated pits

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.     

Brain clathrin: Viscometric and turbidimetric properties of its ultrastructural assemblies

Clathrin was isolated in highly purified form from bovine brain preparations rich in coated vesicles and by some improvements of our previous procedures. At pH 7.5, clathrin's solution was viscous, but clear. At pH 6.5, clathrin's solution was less viscous, but turbid. By electron microscopy, clathrin's turbidity at pH 6.5 correlated with the presence of numerous basket-like lattices or cages; the higher viscosity observed at pH 7.5 correlated with a mixture of various polymeric forms of clathrin having linearly assembled filaments or filamentous bundles of cross-linked clathrin molecules. In vivo, clathrin's capacity for assembling or disassembling itself into baskets or cage-like structures is compatible with a mechanism that retrieves areas of the plasma membrane containing protein molecules, smaller stimulatory or inhibitory compounds bound on the external cell membrane surface.     

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.     

Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins

β-Arrestins play a role in AT₁ endocytosis by binding the cytoplasmic, C-terminus region T332–S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting β-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT₁ to investigate two possibilities: activated AT₁ induces global relocation of β-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract β-arrestins. Results obtained using high osmolarity and dominant-negative β-arrestin confirmed that internalization of AT₁ in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving β-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT₁ internalization. Confocal microscopy revealed that wild-type AT₁ induced a time-dependent translocation of GFP-tagged β-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic β-arrestin 1 at all, and only trafficked β-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both β-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and β-arrestin 1 less so than β-arrestin 2. In conclusion, this study shows that the high affinity binding of β-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced β-arrestin recruitment to the cell surface and AT₁ receptor.     

Leishmania major: clathrin and adaptin complexes of an intra-cellular parasite

To investigate the role of clathrin-mediated trafficking during the Leishmania lifecycle, open reading frames encoding clathrin heavy chain and the beta-adaptins, major components of the adaptor complexes, have been analysed both in silico and experimentally. The Leishmania genome encodes three beta-adaptins, which arose at a time predating speciation of these divergent trypanosomatids. Unlike Trypanosoma brucei, both clathrin heavy chain and beta-adaptin1 are constitutively expressed throughout the Leishmania life cycle. Clathrin relocalises in amastigotes relative to promastigotes, consistent with developmental alterations to the morphology of the endo-membrane system.     

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.     

Trafficking of yellow-fluorescent-protein-tagged mu1 subunit of clathrin adaptor AP-1 complex in living cells

Clathrin adaptor protein AP-1 complex is thought to function in forming clathrin-coated vesicles at the trans -Golgi network (TGN) and mediating transport of cargo between the TGN and endosomes. To study trafficking of AP-1 in living cells, yellow fluorescent protein (YFP) was inserted in the middle of µ1 A subunit of AP-1. When expressed in a tetracycline-dependent manner in HeLa cells, YFP-µ1 was efficiently incorporated into the AP-1 complex, replacing endogenous µ1 in most of cellular AP-1. Time-lapse imaging revealed that YFP-µ1/AP-1 departs from TGN as isolated vesicles and spherical structures, or varicosities, associated with fine tubular processes. Typically, several vesicles or varicosities were seen moving sequentially along the same 'tracks' from TGN to cell periphery. These data suggest that AP-1 may function after formation of Golgi transport intermediates in facilitating their intracellular movement. Mutagenesis of YFP-µ1 determined that the structural requirements for its binding to tyrosine-containing sequence motifs are similar to those previously defined in µ2 subunit of AP-2. Moreover, the carboxyl-terminal half of µ2 could replace the corresponding fragment of µ1 without loss of the ability of the resulting µ1-YFP-µ2 chimeric protein to incorporate into AP-1 and bind tyrosine-containing motifs. Mutations that abolish binding capacity for tyrosine motifs did not mistarget AP-1 in the cell, suggesting that AP-1 interactions with this type of sorting signals are not essential for membrane docking of AP-1 at the TGN. Altogether, this study demonstrates that YFP-tagged µ1 protein can serve as a useful tool for visualizing the dynamics of AP-1 in living cells and for the structure-function analysis of µ1–cargo interactions.     

Lateral forces in AFM imaging and immobilization of cells and organelles

Lateral forces are inevitable in contact mode AFM imaging and they contribute significantly to the image formation under certain conditions. In cases where the objects are comparable in size to the cantilever tip and particularly in cases where the tips have a high aspect ratio, the lateral force may exceed the vertical force and may impose a severe limitation to the stability of the sample during imaging. Here we have calculated the relation between the exerted lateral force and the applied vertical force as a function of the friction coefficient, the geometry of the tip, and the stiffness of the cantilever. We present a strategy to immobilize larger particles by sucking them into the pores of nucleopore filters and binding them by chemical cross linking. High resolution images of nematocysts which were immobilized with this strategy are presented. The images reveal the supra-molecular arrangement of the mini-collagen of the capsule wall. Received: 11 March 1996 / Accepted: 23 April 1997     

Preferential insertion of lactose permease in phospholipid domains: AFM observations

We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8 nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.