Domain structure of yeast and human amphiphysin family proteins. Schematic representation of the domain organization of the budding yeast amphiphysin family proteins Rvs161p and Rvs167p, the fission yeast amphiphysin family proteins Hob1 and Hob3, and the human amphiphysin family proteins amphiphysin 1, Bin1/amphiphysin 2 (several tissue-specific and ubiquitous splice variants), Bin2, and Bin3, and endophilins A1, A2, A3, B1, and B2.

Subcellular localization of Rvs167p in growing and mating yeast cells. Shown are budding yeast cells that express a fusion protein (Rvs167p-GFP) that comprises full-length Rvs167p with GFP inserted between the BAR and GPA-rich domains of Rvs167p. The same fields of cells were viewed by fluorescence optics (right) to visualize GFP and by differential interference contrast (left) to visualize the cell profiles. A, unbudded cell; B, cell at an early stage of bud emergence; C, cell with small bud; D, cell with large bud; E, cells undergoing division (cytokinesis); F, cells arrested in G₁ and forming a mating projection (shmoo) after pheromone treatment (only one cell expressing Rvs167p-GFP is depicted in this panel). In vegetatively growing cells Rvs167p-GFP localizes to large cortical patches that polarize to nascent bud sites, small buds, and the bud neck during cell division (cytokinesis). In mating cells Rvs167p-GFP concentrates at the tip of the mating projection (shmoo). (Reprinted from reference 11 with permission of the Company of Biologists Ltd.)

Ultrastructure of wild-type, fus2, rvs161, and rvs161 fus2 cells during cell-cell fusion. Shown are electron micrographs of wild-type (WT), fus2Δ, rvs161Δ, and rvs161Δ fus2Δ cells undergoing cell-cell fusion during mating. Note that wild-type cells exhibit restricted zones of cell-cell contact during fusion while fus2Δ, rvs161Δ, and rvs161Δ fus2Δ mutants exhibit very wide zones of cell-cell contact during fusion. Wild-type and mutant cells all exhibit numerous vesicles that cluster at the zone of cell-cell fusion. Note that plasma membrane invaginations/tubules accumulate at the zone of fusion in the mutant cells, but are less common in wild-type cells. Labels indicate plasma membrane invaginations (black arrows), vesicle clusters (black arrowheads), electron-dense plaques (white arrows), and nonremoved cell wall remnants (asterisks). Bars = 1 μm. (These previously unpublished images are shown here with the kind permission of Alison Gammie.)

Defects in vesicle fusion in rvs161 mutant cells result in vesicle accumulation. Shown are electron micrographs of wild-type (A), rvs161Δ (B), and rvs167Δ (C) yeast cells. The panels on the left depict cells grown under standard conditions,while those on the right depict cells grown in medium containing sublethal levels (3.4%, wt/vol) of Na⁺. Bar, 1 μm. Labeled organelles: n, nucleus; v, vacuole; vs, vesicles; g, Golgi apparatus apparatus; ps, primary septum; s, septum. The arrow indicates cell wall abnormalities. (Reproduced from reference 20 with permission of the publisher. Copyright John Wiley and Sons Ltd.)

Functional classification of Rvs161p and Rvs167p-interacting proteins. Pie chart depicting the proportions of the total set of known Rvs161p- and Rvs167p-interacting proteins (including both physical and genetic) which belong to each functional category.

Amphiphysin 1 evaginates liposomes in vitro to generate coated membrane tubules. A, electron micrographs of negatively stained membrane tubules generated in vitro from spherical liposomes composed of brain lipids a) after incubation with recombinant full-length amphiphysin 1 (cleaved from a GST fusion protein) or b) after incubation with a recombinant fusion protein comprising GST and the amphiphysin 1 BAR domain only (residues 1 to 286). Bar, 500 nm. B, electron micrographs of negatively stained membrane tubules generated in vitro from spherical liposomes composed of brain lipids after incubation with clathrin coat components plus: a, recombinant dynamin 1; b, both recombinant dynamin 1 and recombinant amphiphysin 1; or c, recombinant amphiphysin 1. The insets at the top right of each panel show the ends of tubules at high magnification (a, negatively stained sample; b and c, positively stained embedded and thin-sectioned samples). For comparison, the bottom inset in panel a shows clathrin-coated buds at high magnification. Note that the presence of amphiphysin 1 in panels b and c increases the frequency of clathrin-coated bud formation at tubule ends compared to dynamin 1 alone in panel a. C, electron micrographs showing the appearance of the tubular coat that forms when spherical liposomes are incubated in vitro with recombinant amphiphysin 1 alone (a and b); recombinant dynamin 1 alone (c and d); both recombinant amphiphysin 1 and recombinant dynamin 1 (e and f), or total brain cytosol with ATP and GTPγS (g). The images in panels a, c, and e represent negatively stained samples, while those in panels b, d, f, and g represent samples embedded, thin sectioned, and then positively stained. Note that recombinant amphiphysin 1 or dynamin 1 alone forms thin tightly packed rings on membrane tubules while recombinant amphiphysin 1 and dynamin 1 together or endogenous amphiphysin 1 and dynamin 1 in total brain extract form thick rings with regular spacing on membrane tubules. (Reprinted from reference 316 with permission of the publisher.)

Three-dimensional crystal structure of the Drosophila amphiphysin BAR domain. A, Two amphiphysin monomers (depicted in purple and green) associate in antiparallel orientation to form a banana-shaped homodimer represented here in ribbon format. The residues that form the basic patches on the concave membrane-binding surface of the dimer are highlighted. The Protein Data Bank identifier is 1URU. Note that the Drosophila amphiphysin BAR domain lacks an NTID, but sequence alignment with mammalian Bin1 suggests the NTID would be positioned within the unstructured loop between helices 2 and 3 as seen in the Drosophila BAR domain. This loop is positively charged and two conserved lysine residues have been shown to be important for membrane association of both Drosophila amphiphysin and rat amphiphysins 1 and 2 (237). The presence of the NTID would contribute extra positive charges to the unstructured loop and may further strengthen membrane association. B, The homodimer represented in electrostatic surface format and viewed with the same orientation as in A. Electrostatic potential is indicated: −10 kTe⁻¹ in red; +10 kTe⁻¹ in blue). C, The homodimer represented in ribbon format but viewed at an angle perpendicular to that in panel A to highlight details of the concave face. D, The homodimer represented in electrostatic surface format and viewed with the same orientation as in panel C. (Reprinted from reference 237 with permission of the publisher. Copyright 2004 American Association for the Advancement of Science.)

Subcellular localization of Rvs161p in growing and mating yeast cells. A. Shown are budding yeast cells that express fusion proteins that comprise full-length Rvs161p fused to the fluorescent reporter Aequoria victoria green fluorescent protein (GFP). The same fields of cells were viewed by fluorescence optics (left) to visualize GFP and by differential interference contrast optics (right) to visualize the cell profiles. In panels A to J, M, and N the cells express Rvs161p-GFP with the reporter fused at the Rvs161pC terminus. In panels K and L the cells express GFP-Rvs161p with the reporter fused at the Rvs161pN terminus. Panels A to F show vegetatively growing cells, panels G and H show cells arrested in G₁ and forming mating projections after pheromone treatment, and panels I to N show mating cells. In pheromone-treated cells Rvs161p-GFP concentrates at the tip of the mating projection (shmoo) and in mating cells Rvs161p-GFP and GFP-Rvs161p concentrate at the site of cell-cell fusion. In vegetatively growing cells Rvs161p-GFP exhibits a diffuse cytoplasmic distribution and is excluded from the vacuole (large indentation apparent in the cell profiles). (Reproduced from reference 21 by copyright permission of The Rockefeller University Press.) B. Shown are vegetatively growing budding yeast cells that express a fusion protein (Rvs161p-GFP) that comprises full-length Rvs161p with GFP fused at the Rvs161pC terminus. The same fields of cells were viewed by fluorescence optics (center and right columns) to visualize GFP and by differential interference contrast (left column) to visualize the cell profiles. For each field of cells two focal planes were viewed by fluorescence optics: an equatorial view (center column) and a top view (right column). A, an unbudded cell; B, a cell at an early stage of bud emergence; C, cells undergoing cell division (cytokinesis). This Rvs161p-GFP fusion protein localizes to numerous small cortical patches. (Reprinted with permission from reference 10.)

Rvs161p functions in cell-cell fusion during mating. Shown are matings between two wild-type haploids (panels A and B) or between two rvs161Δ mutant haploids (panels C to F) visualized by fluorescence/differential interference contrast optics. Cell nuclei are stained with the fluorescent dye 4′,6′-diamidino-2-phenylindole. Cell-cell fusion allows cytoplasmic mixing and the nuclei of each mating partner meet at the site of cell fusion to fuse and create a single diploid nucleus. In the case of rvs161Δ × rvs161Δ matings, delays in cell-cell fusion (apparent by differential interference contrast) result in delays in nuclear fusion. This leads to the persistence of two nuclei. In panels C and D nuclear fusion is taking place despite incomplete removal of the intervening cell wall (partial Fus⁻ phenotype) and in panels E and F nuclear fusion has been completely blocked by the inability to remove intervening cell wall (full Fus⁻ phenotype). (Reproduced from reference 21 by copyright permission of the Rockefeller University Press.)

Ultrastructure of wild-type, rvs161Δ, and rvs167Δ cells during cell-cell fusion. Shown are electron micrographs of wild-type, rvs161Δ, and rvs167Δ cells undergoing cell-cell fusion during mating. Panels a to c, matings between two wild-type cells; panels d to f, matings between two rvs161Δ cells; panels g and h, matings between two rvs167Δ cells. Bar, 1 μm. Labeled organelles: n, nucleus; v, vacuole; vs, vesicles; g, Golgi apparatus. (Reproduced from reference 20 with permission of the publisher. Copyright John Wiley and Sons Ltd.)

Sphingolipid biosynthesis in yeast. The yeast sphingolipid biosynthetic pathway indicating the steps blocked by sur1, sur2, sur4, fen1/sur5, and ipt1 mutations that suppress the phenotypes of rvs161 and rvs167 mutants. CoA, coenzyme A; LCB, long-chain base; PI, phosphatidylinositol; Inos-P, inositol-1-P; Cer, ceramide; IPC, inositolphosphorylceramide; MIPC, mannosyl-inositolphoshorylceramide; M(IP)₂C, mannosyl-diinositolphoshorylceramide; Man-P-Inos, mannose(α-1,2)inositol-1-P. (Reprinted from reference 54 with permission from Elsevier.)

Amphiphysin 1 SH3 domain interactions are required for fission of endocytic vesicles. Lamprey giant reticulospinal synapses were injected with a fusion protein comprising glutathione S-transferase and the human amphiphysin 1 SH3 domain (GST-amphSH3) and effects on synaptic vesicle recycling from the plasmalemma (plasma membrane) to regenerate the cytoplasmic vesicle pool were examined. Panel A, electron micrograph of a synapse in an axon injected with GST-amphSH3 and then maintained in low Ca²⁺ (0.1 mM Ca²⁺, 4 mM Mg²⁺), without stimulation. Panel B, electron micrograph of a synapse in an axon injected with GST-amphSH3 and then electrically stimulated at 0.2 Hz for 30 min in high Ca²⁺ (2.6 mM Ca²⁺ and 1.8 mM Mg²⁺) to induce exocytosis of synaptic vesicles. Panel C, electron micrograph of a synapse in an axon treated as in panel B showing the accumulation of invaginated endocytic pits at the plasmalemma. Panel D, electron micrograph of a synapse in an axon injected with GST-amphSH3 and then electrically stimulated at 5 Hz for 30 min in high Ca²⁺ (2.6 mM Ca²⁺ and 1.8 mM Mg²⁺) to induce exocytosis of synaptic vesicles. Bars, 0.2 μm. The bar in panel A applies to panels A to C. (Reprinted from reference 293 with permission of the publisher. Copyright 1997 American Association for the Advancement of Science.)

Bin1−10−12−13 functions in phagocytosis in macrophages. RAW-TT10 (murine macrophage-derived) cells were transiently cotransfected with a construct that expresses GFP and with either an empty vector (Control) or a construct that expresses a dominant negative form of Bin1−10−12−13 lacking the C-terminal SH3 domain (AmphIImSH3⁻). The cells expressing the highest levels of GFP were recovered by flow cytometry and incubated with immunoglobulin G-coated sheep red blood cells for 10 min (panels A and B) or for 1 h (panels C and D) before visualization by scanning electron microscopy. The control cells formed actin pedestals beneath the bound sheep red blood cells, then formed membrane ruffles (A), and then phagocytosed the bound sheep red blood cells (C). In contrast, cells expressing the dominant negative mutant Bin1−10−12−13 lacking the C-terminal SH3 domain formed actin pedestals beneath the bound sheep red blood cells (B), but did not form membrane ruffles, and did not phagocytose the bound sheep red blood cells which remained on the cell surface (D). The scale bar in panel B applies to each panel. (Reprinted from reference 100 with permission from Elsevier.)