Dynamics of clathrin and adaptor proteins during endocytosis

2006 ◽  
Vol 291 (5) ◽  
pp. C1072-C1081 ◽  
Author(s):  
Joshua Z. Rappoport ◽  
Shahrnaz Kemal ◽  
Alexandre Benmerah ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Time Course ◽  
Adaptor Proteins ◽  
Nucleation Site ◽  
Heterogeneous Population ◽  
Coated Vesicles ◽  
Subunit Exchange ◽  
Dead End ◽  
Endocytic Vesicles

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 ≥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.

scholarly journals The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

1991 ◽  
Vol 113 (4) ◽  
pp. 731-741 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Coated Vesicles ◽  
Minor Role ◽  
Specific Marker ◽  
Con A ◽  
Early Endosomes ◽  
Gold Conjugate ◽  
Entire Cell ◽  
A Minor ◽  
Endocytic Vesicles

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

scholarly journals ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

1998 ◽  
Vol 9 (8) ◽  
pp. 2217-2229 ◽  
Author(s):  
Lisa A. Hannan ◽  
Sherri L. Newmyer ◽  
Sandra L. Schmid
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Dual Role ◽  
Vesicular Trafficking ◽  
Coated Vesicles ◽  
Golgi Network ◽  
Cytosolic Factor

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

scholarly journals A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells

1994 ◽  
Vol 124 (5) ◽  
pp. 667-675 ◽  
Author(s):  
P Thomas ◽  
AK Lee ◽  
JG Wong ◽  
W Almers
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Time Constant ◽  
Flash Photolysis ◽  
Membrane Capacitance ◽  
Coated Vesicles ◽  
Neuroendocrine Cells ◽  
Pituitary Cells ◽  
Excitable Cells ◽  
Late Step

In neuroendocrine cells, cytosolic Ca2+ triggers exocytosis in tens of milliseconds, yet known pathways of endocytic membrane retrieval take minutes. To test for faster retrieval mechanisms, we have triggered short bursts of exocytosis by flash photolysis of caged Ca2+, and have tracked subsequent retrieval by measuring the plasma membrane capacitance. We find that a limited amount of membrane can be retrieved with a time constant of 4 s at 21-26 degrees C, and that this occurs partially via structures larger than coated vesicles. This novel mechanism may be arrested at a late step. Incomplete retrieval structures then remain on the cell surface for minutes until the consequences of a renewed increase in cytosolic [Ca2+] disconnect them from the cell surface in < 1 s. Our results provide evidence for a rapid, triggered membrane retrieval pathway in excitable cells.

scholarly journals alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

1979 ◽  
Vol 82 (3) ◽  
pp. 614-625 ◽  
Author(s):  
M C Willingham ◽  
F R Maxfield ◽  
I H Pastan
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Coated Vesicles ◽  
The Other ◽  
Con A ◽  
Coated Pits ◽  
Cultured Fibroblasts ◽  
Receptor Complexes ◽  
Transmission Electron ◽  
Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

scholarly journals Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison.

1981 ◽  
Vol 29 (12) ◽  
pp. 1437-1441 ◽  
Author(s):  
P F Davies ◽  
L Kuczera
Keyword(s):  
Cell Surface ◽  
Vascular Endothelium ◽  
Ruthenium Red ◽  
Coated Vesicles ◽  
Membrane Glycoproteins ◽  
Coated Pits ◽  
Ruthenium Red Staining ◽  
Almost All ◽  
Endocytic Vesicles

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.

scholarly journals Vps10p Cycles between the TGN and the Late Endosome via the Plasma Membrane in Clathrin Mutants

2002 ◽  
Vol 13 (12) ◽  
pp. 4296-4307 ◽  
Author(s):  
Olivier Deloche ◽  
Randy W. Schekman
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Late Endosome ◽  
Coated Vesicles ◽  
Cargo Protein ◽  
Endosomal Membranes ◽  
Green Fluorescent ◽  
Vacuolar Protein

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in achc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport inchc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, inchc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normalVPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.

scholarly journals Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Madhumitha Narasimhan ◽  
Alexander Johnson ◽  
Roshan Prizak ◽  
Walter Anton Kaufmann ◽  
Shutang Tan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Constant Curvature ◽  
Imaging Techniques ◽  
Live Imaging ◽  
Unique Role ◽  
Major Route ◽  
Bona Fide ◽  
Endocytic Vesicles

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

scholarly journals Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells

1991 ◽  
Vol 278 (1) ◽  
pp. 235-241 ◽  
Author(s):  
A E Clark ◽  
G D Holman ◽  
I J Kozka
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glucose Transport ◽  
Time Course ◽  
Transport Activity ◽  
Insulin Stimulation ◽  
Surface Labelling ◽  
Lag Period ◽  
Adipose Cells ◽  
Stimulation Of

We have used an impermeant bis-mannose compound (2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos+ ++- 4-yloxy)-2- propylamine; ATB-BMPA) to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18 degrees C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t 1/2 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t 1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or less of cell-surface transporters over 60 min at 18 degrees C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Intracellular galectins control cellular responses commensurate with cell surface carbohydrate composition

Glycobiology ◽  
2019 ◽  
Vol 30 (1) ◽  
pp. 36-48 ◽  
Author(s):  
Ming-Hsiang Hong ◽  
Wei-Han Lin ◽  
I-Chun Weng ◽  
Yu-Hsien Hung ◽  
Hung-Lin Chen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Super Resolution ◽  
Adaptor Proteins ◽  
Cellular Responses ◽  
Cell Surface Carbohydrate ◽  
Galectin 3 ◽  
Carbohydrate Ligands ◽  
Resolution Imaging ◽  
Endocytic Vesicles ◽  
Cell Surface Glycans

Abstract Galectins are β-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans.

scholarly journals GLUT4 trafficking in insulin-stimulated rat adipose cells: evidence that heterotrimeric GTP-binding proteins regulate the fusion of docked GLUT4-containing vesicles

1999 ◽  
Vol 343 (3) ◽  
pp. 571-577 ◽  
Author(s):  
Cynthia M. FERRARA ◽  
Samuel W. CUSHMAN
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Adenosine Deaminase ◽  
Glucose Transport ◽  
Time Course ◽  
Transport Activity ◽  
Experimental Conditions ◽  
Distinct Sequence ◽  
Kinetics Of ◽  
Adipose Cells

Agents that activate the G-protein Gi (e.g. adenosine) increase, and agents that activate Gs [e.g. isoprenaline (isoproterenol)] decrease, steady-state insulin-stimulated glucose transport activity and cell-surface GLUT4 in isolated rat adipose cells without changing plasma membrane GLUT4 content. Here we have further examined the effects of RsGs and RiGi ligands (in which Rs and Ri are Gs- and Gi-coupled receptors respectively) on insulin-stimulated cell-surface GLUT4 and the kinetics of GLUT4 trafficking in these same cells. Rat adipose cells were preincubated for 2 min with or without isoprenaline (200 nM) and adenosine deaminase (1 unit/ml), to stimulate Gs and decrease the stimulation of Gi respectively, followed by 0-20 min with insulin (670 nM). Treatment with isoprenaline and adenosine deaminase decreased insulin-stimulated glucose transport activity by 58%. Treatment with isoprenaline and adenosine deaminase also resulted in similar decreases in insulin-stimulated cell-surface GLUT4 as assessed by both bis-mannose photolabelling of the substrate-binding site and biotinylation of the extracellular carbohydrate moiety when evaluated under similar experimental conditions. After stimulation with insulin in the absence of Gs and the presence of Gi agents, a distinct sequence of plasma membrane events took place, starting with an increase in immunodetectable GLUT4, then an increase in the accessibility of GLUT4 to bis-mannose photolabel, and finally an increase in glucose transport activity. Pretreatment with isoprenaline and adenosine deaminase before stimulation with insulin did not affect the time course of the increase in immunodetectable GLUT4 in the plasma membrane, but did delay both the increase in accessibility of GLUT4 to photolabel and the increase in glucose transport activity. These results suggest that RsGs and RiGi modulate insulin-stimulated glucose transport by influencing the extent to which GLUT4 is associated with occluded vesicles attached to the plasma membrane during exocytosis, perhaps by regulating the fusion process through which the GLUT4 in docked vesicles becomes exposed on the cell surface.

Sign in / Sign up

Export Citation Format

Share Document