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.

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 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.

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.

scholarly journals The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.

1996 ◽  
Vol 133 (6) ◽  
pp. 1237-1250 ◽  
Author(s):  
K Takei ◽  
O Mundigl ◽  
L Daniell ◽  
P De Camilli
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Nerve Terminals ◽  
Vesicle Budding ◽  
Vesicle Cycle ◽  
Single Vesicle

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.

scholarly journals Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

1993 ◽  
Vol 120 (1) ◽  
pp. 37-45 ◽  
Author(s):  
L L Carter ◽  
T E Redelmeier ◽  
L A Woollenweber ◽  
S L Schmid
Keyword(s):  
Protein Function ◽  
Binding Proteins ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Gtp Binding Proteins ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding ◽  
Gamma S

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

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.

Self-Assembly of Dynamin

2001 ◽  
Vol 7 (S2) ◽  
pp. 1210-1211
Author(s):  
Dganit Danino ◽  
Jenny E. Hinshaw
Keyword(s):  
Plasma Membrane ◽  
Self Assembly ◽  
Wild Type ◽  
Salt Solutions ◽  
Coated Pits ◽  
Vesicle Recycling ◽  
Rich Domain ◽  
Gtp Binding ◽  
Low Salt ◽  
Over Time

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

scholarly journals Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.

1992 ◽  
Vol 119 (5) ◽  
pp. 1163-1171 ◽  
Author(s):  
E Smythe ◽  
L L Carter ◽  
S L Schmid
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicle ◽  
A431 Cells ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Membrane Fission ◽  
Cytosolic Factors ◽  
Insight Into ◽  
Stimulation Of

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.

scholarly journals Mutations in human dynamin block an intermediate stage in coated vesicle formation

1993 ◽  
Vol 122 (3) ◽  
pp. 553-563 ◽  
Author(s):  
AM van der Bliek ◽  
TE Redelmeier ◽  
H Damke ◽  
EJ Tisdale ◽  
EM Meyerowitz ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Mammalian Cells ◽  
Intermediate Stage ◽  
Binding Domain ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding

The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.

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