scholarly journals Dynamin GTPase Domain Mutants Block Endocytic Vesicle Formation at Morphologically Distinct Stages

2001 ◽  
Vol 12 (9) ◽  
pp. 2578-2589 ◽  
Author(s):  
Hanna Damke ◽  
Derk D. Binns ◽  
Hideho Ueda ◽  
Sandra L. Schmid ◽  
Takeshi Baba
Keyword(s):  
Dominant Negative ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Complex Relationship ◽  
Protein Activity ◽  
Coated Pits ◽  
Gtpase Domain ◽  
Receptor Mediated Endocytosis ◽  
Gtpase Cycle

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.

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 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 Reconstitution of clathrin-coated pit budding from plasma membranes.

1991 ◽  
Vol 114 (5) ◽  
pp. 881-891 ◽  
Author(s):  
H C Lin ◽  
M S Moore ◽  
D A Sanan ◽  
R G Anderson
Keyword(s):  
Plasma Membranes ◽  
Ldl Receptor ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gold Label ◽  
Cytosolic Factors

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.

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.

scholarly journals AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

1998 ◽  
Vol 140 (5) ◽  
pp. 1055-1062 ◽  
Author(s):  
Alexandre Benmerah ◽  
Christophe Lamaze ◽  
Bernadette Bègue ◽  
Sandra L. Schmid ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Coated Vesicle ◽  
Mutant Form ◽  
A431 Cells ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

scholarly journals Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease

2016 ◽  
Vol 9 (423) ◽  
pp. ra37-ra37 ◽  
Author(s):  
Arthur Marivin ◽  
Anthony Leyme ◽  
Kshitij Parag-Sharma ◽  
Vincent DiGiacomo ◽  
Anthony Y. Cheung ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Neural Crest Cell ◽  
Craniofacial Development ◽  
Dominant Negative ◽  
Protein Subunit ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Heterotrimeric G Protein

Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein–coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ETAR), which is mediated by the G protein (heterotrimeric guanine nucleotide–binding protein) subunit Gαq/11 and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations in GNAI3, which encodes Gαi3, but it is unknown whether this G protein has a role within the ETAR pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gαi3 mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gαi3 mutant proteins that couple to ETAR but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gαq/11 and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.

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.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

2000 ◽  
Vol 113 (11) ◽  
pp. 1993-2002 ◽  
Author(s):  
H. Cao ◽  
H.M. Thompson ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Living Cells ◽  
Dominant Negative ◽  
Coated Vesicle ◽  
Large Numbers ◽  
Golgi Structure ◽  
And Function ◽  
Golgi Network ◽  
Mutant Cells

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

Cellular Mechanisms For Transferrin Receptor Endocytosis Vital For Terminal Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3434-3434
Author(s):  
Yuichi Ishikawa ◽  
Silvia K Tacheva-Grigorova ◽  
Manami Maeda ◽  
Francois Aguet ◽  
Sung-Uk Lee ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Erythroid Differentiation ◽  
Live Cell ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Membrane Tension ◽  
Erythroid Development

Abstract Clathrin-mediated endocytosis (CME) is a key endocytic portal that regulates transferrin receptor (TfR) endocytosis. Although CME is among the most-studied cellular systems in cell biology, little is known regarding how this key system physiologically functions in vivo.The CALM gene (for Clathrin Assembly Lymphoid Myeloid, also known as PICALM), located on human chromosome 11q14, encodes a 652 aa protein containing multiple domains functioning in clathrin-coated vesicle formation. Since CALM is predominantly expressed in erythroblasts, and hematopoietic-specific Calmconditional knockout mice, which we developed, exhibit a severe iron-deficient anemia, we hypothesized that CALM functions to increase efficiency of TfR endocytosis in erythroblasts so that iron uptake is not rate-limiting. To test this hypothesis, we performed FACS-based endocytosis assay in primary erythroblasts, in vitro erythroid differentiation assay, live-cell imaging and CALM add-back rescue experiments. TfR endocytosis was severely attenuated in Calm-deficient basophilic- and polychromatophilic erythroblasts: efficiency of TfR endocytosis was less than 25% of that of control, as revealed by FACS-based endocytosis assay. Unexpectedly, TfR endocytosis was not active in orthochromatophilic erythroblasts regardless of genotype, while they express TfR at high levels.For live cell imaging, we established immortalized mouse embryonic fibroblasts (MEFs), in which the Calm gene can be deleted in an inducible manner (CalmF/F ERT2-Cre+ MEFs). The MEFs were then stably transduced with the retrovirus encoding an EGFP-tagged AP2-bound adaptin σ2. The dynamics of clathrin-coated vesicle formation was determined using fast live cell fluorescence spinning disk confocal microscopy. Clathrin-coat dynamics was barely affected in the absence of Calm under regular culture condition; however, it was completely stalled under high membrane tension (e.g. hypo-osmotic medium, jasplakinolide treatment). To determine functional domain(s) of CALM, a series of CALM mutants were generated, “added-back” to Calm-deficient MEFs and coat dynamics examined by live cell imaging. Mutant that does not bind to phosphatidylinositol 4,5-bisphosphate of the plasma membrane (PIP2-mutant CALM) failed to rescue TfR endocytosis in Calm-deficient MEFs, suggesting that CALM binding to PIP2, but not to v-SNAREs or EPS15, is essential for CALM-mediated TfR endocytosis. Splenic erythroblasts were significantly expanded in Calm conditional knockout mice. In contrast, Calm-deficient hematopoietic progenitors cannot give rise to immature erythroblasts in vitro, suggesting that Calm-deficiency is partially compensated in vivo via non-cell autonomous mechanisms (e.g. central macrophages). Iron supplement treatment could significantly rescue erythroid development of Calm-deficient progenitors in vitro, indicating that iron-deficiency is a primary cause of developmental defects seen in Calm-deficient erythroblasts. As expected, retrovirus-mediated expression of WT-Calm, but not PIP2-mutant, completely rescued erythroid development in Calm-deficient erythroblasts in vitro.Our data indicate that CALM plays a key role in clathrin-mediated endocytosis in a context-dependent (high membrane tension) and cell-type-specific (erythroblasts) manner. We propose that CALM is essential for transferrin uptake in erythroblasts by functioning as an erythroid-specific clathrin adaptor. Disclosures: No relevant conflicts of interest to declare.

Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation

2003 ◽  
Vol 31 (3) ◽  
pp. 736-739 ◽  
Author(s):  
E. Smythe
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Coated Pits ◽  
Permeabilized Cell ◽  
Cytosolic Proteins ◽  
Cell Assays

Clathrin-coated pits are the major ports of entry into the cell and are responsible for the internalization of a variety of biologically important macromolecules. These transport intermediates form as a result of the co-ordinated assembly of a number of cytosolic proteins on to the membrane which results in specific cargo recruitment. We have used a variety of approaches including permeabilized cell assays and light and electron microscopy to identify and characterize the proteins and enzymes involved in coated vesicle formation.

Sign in / Sign up

Export Citation Format

Share Document