scholarly journals The Role of Dynamin and Its Binding Partners in Coated Pit Invagination and Scission

2001 ◽  
Vol 152 (2) ◽  
pp. 309-324 ◽  
Author(s):  
Elaine Hill ◽  
Jeroen van der Kaay ◽  
C. Peter Downes ◽  
Elizabeth Smythe
Keyword(s):  
Plasma Membrane ◽  
Sh3 Domain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Binding Partners ◽  
Late Stages

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.

scholarly journals Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.

1994 ◽  
Vol 127 (4) ◽  
pp. 915-934 ◽  
Author(s):  
H Damke ◽  
T Baba ◽  
D E Warnock ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Wild Type ◽  
Coated Pits ◽  
Bulk Fluid ◽  
Vesicle Budding ◽  
Gtp Binding ◽  
Hela Cell Lines

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

Regulation of clathrin-coated vesicle formation

2001 ◽  
Vol 29 (4) ◽  
pp. 375-377 ◽  
Author(s):  
E. Hill ◽  
O. Olusanya ◽  
J. van der Kaay ◽  
C. P. Downes ◽  
P. D. Andrews ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coat Proteins ◽  
Concerted Action ◽  
Coated Pits ◽  
The Real ◽  
Permeabilized Cells ◽  
Real Challenge

The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.

scholarly journals Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transport: evidence for coated vesicle-independent transport.

1994 ◽  
Vol 5 (2) ◽  
pp. 237-252 ◽  
Author(s):  
T C Taylor ◽  
M Kanstein ◽  
P Weidman ◽  
P Melançon
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Specific Inhibitor ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Golgi Transport ◽  
Independent Mechanism ◽  
Free Transport

We investigated the role of ADP-ribosylation factors (ARFs) in Golgi function using biochemical and morphological cell-free assays. An ARF-free cytosol produced from soluble Chinese hamster ovary (CHO) extracts supports intra-Golgi transport by a mechanism that is biochemically indistinguishable from control transport reactions: ARF-free transport reactions are NSF-dependent, remain sensitive to the donor Golgi-specific inhibitor ilimaquinone, and exhibit kinetics that are identical to that of control reactions containing ARFs. In contrast, ARF-free cytosol does not support the formation of coated vesicles on Golgi cisternae. However, vesicle formation is reconstituted upon the addition of ARF1. These data suggest that neither soluble ARFs nor coated vesicle formation are essential for transport. We conclude that cell-free intra-Golgi transport proceeds via a coated vesicle-independent mechanism regardless of vesicle formation on Golgi cisternae.

scholarly journals Immunofluorescent localization of 100K coated vesicle proteins.

1986 ◽  
Vol 102 (1) ◽  
pp. 48-54 ◽  
Author(s):  
M S Robinson ◽  
B M Pearse
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Limited Proteolysis ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Cell Protein ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Double Labeling ◽  
Vesicle Proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

scholarly journals Three-dimensional visualization of coated vesicle formation in fibroblasts.

1980 ◽  
Vol 84 (3) ◽  
pp. 560-583 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Receptor Mediated Endocytosis ◽  
Replication Method ◽  
Complete Contraction ◽  
Partial Contraction ◽  
Selective Mechanism

Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep-etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.

Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin

2001 ◽  
Vol 114 (2) ◽  
pp. 353-365 ◽  
Author(s):  
X. Zhao ◽  
T. Greener ◽  
H. Al-Hasani ◽  
S.W. Cushman ◽  
E. Eisenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Coated Vesicles ◽  
Coated Pits ◽  
Trans Golgi Network ◽  
J Domain ◽  
Almost All ◽  
Golgi Network ◽  
Assembly Proteins

Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of auxilin indeed inhibited endocytosis. However, expression of auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.

scholarly journals AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

2009 ◽  
Vol 20 (20) ◽  
pp. 4278-4288 ◽  
Author(s):  
Yujia Wen ◽  
Irene Stavrou ◽  
Kirill Bersuker ◽  
Rebecca J. Brady ◽  
Arturo De Lozanne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Contractile Vacuole ◽  
Snare Proteins ◽  
In Vitro Assays ◽  
Accessory Proteins ◽  
Coated Pits ◽  
Contractile Vacuoles ◽  
Null Mutants

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

scholarly journals Clathrin-mediated endocytosis in AP-2–depleted cells

2003 ◽  
Vol 162 (5) ◽  
pp. 909-918 ◽  
Author(s):  
Alison Motley ◽  
Nicholas A. Bright ◽  
Matthew N.J. Seaman ◽  
Margaret S. Robinson
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Ldl Receptor ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Golgi Membrane ◽  
Coated Pits ◽  
Ldl Receptors ◽  
Cargo Proteins ◽  
Membrane Compartments

We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

scholarly journals Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes.

1987 ◽  
Vol 105 (1) ◽  
pp. 199-206 ◽  
Author(s):  
P J DiMario ◽  
A P Mahowald
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Activity ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Class Analysis ◽  
Wild Type ◽  
Coated Pits ◽  
Protein Precursor ◽  
Sterile Mutant

Ultrastructural analysis of developing oocytes produced by the recessive female sterile mutant, yolkless (yl), in Drosophila melanogaster shows that yl+ gene activity is necessary for coated pit and coated vesicle formation within these oocytes. 29 alleles of the mutation are known to exist, and they fall either within a strongly affected class or a weakly affected class. Analysis of oocytes produced by females homozygous for the strongly affected class of alleles shows a greater than 90% reduction in the numbers of coated pits and coated vesicles. These oocytes have very little proteinaceous yolk, and the females accumulate vitellogenin (the yolk protein precursor) within their hemolymph. Moreover, females homozygous or hemizygous for a given strong allele produce mature oocytes that are flaccid. Alternatively, females homozygous or hemizygous for weak alleles produce yolk-filled oocytes, but the number of coated pits and coated vesicles within these oocytes is 50% of that found in the oocytes of wild-type females. Despite the presence of yolk within these oocytes, females homozygous for weak yl- alleles remain sterile, and their mature oviposited eggs collapse with time.

Structural Studies of Dynamin Tubular Crystals by Cryo-Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 1024-1025
Author(s):  
Peijun Zhang ◽  
Jenny E. Hinshaw
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Lipid Vesicles ◽  
Coated Vesicle ◽  
Permissive Temperature ◽  
Coated Pits ◽  
Unique Role ◽  
Cryo Electron Microscopy ◽  
Tubular Crystals

Dynamin is a 100 kD GTPase that plays an essential role in clathrin-coated vesicle formation during receptor mediated endocytosis, and in caveolae internalization and may play a role in intracellular membrane trafficking (1). It shares an extensive sequence homology (70% identity) to shibiregene product in Drosophila(2,3). The shibiretsmutants exhibit a rapid and reversible paralysis at non-permissive temperature due to a depletion of synaptic vesicles in their nerve termini which is believed to be caused by a block in endocytosis since there is an accumulation of “collared” clathrin-coated pits at the plasma membrane (4). Synaptosomes treated with GTPγs produces elongated necks surrounded by dynamin (6). Purified recombinant dynamin itself can assemble to form spirals and bind to lipid vesicles to form tubes, which resemble the “collar” at the necks of coated pits (5). These dynamin tubes vesiculate upon GTP treatment (7), suggesting a unique role of dynamin acting as a mechanoenzyme which causes clathrin-coated vesicles to be pinched off plasma membrane.

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