scholarly journals FUS1Regulates the Opening and Expansion of Fusion Pores between Mating Yeast

2006 ◽  
Vol 17 (5) ◽  
pp. 2439-2450 ◽  
Author(s):  
Scott Nolan ◽  
Ann E. Cowan ◽  
Dennis E. Koppel ◽  
Hui Jin ◽  
Eric Grote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Yeast Cells ◽  
Fusion Pore ◽  
Sudden Increase ◽  
Fluorescent Markers ◽  
Fold Reduction ◽  
Fusion Pores

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.

scholarly journals Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

2021 ◽  
Author(s):  
Olivia Muriel ◽  
Laetitia Michon ◽  
Wanda Kukulski ◽  
Sophie G Martin
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Sexual Reproduction ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Turgor Pressure ◽  
Wall Material ◽  
M Cells ◽  
P Cells ◽  
Cell Cell

Cell-cell fusion is central to the process of fertilization for sexual reproduction. This necessitates the remodeling of peri-cellular matrix or cell wall material and the merging of plasma membranes. In walled fission yeast S. pombe, the fusion of P and M cells during sexual reproduction relies on the fusion focus, an actin structure that concentrates glucanase-containing secretory vesicles for local cell wall digestion necessary for membrane fusion. Here, we present a correlative light and electron microscopy (CLEM) quantitative study of a large dataset of 3D tomograms of the fusion site, which revealed the ultrastructure of the fusion focus as an actin-containing, vesicle-dense structure excluding other organelles. Unexpectedly, the data revealed asymmetries between the two gametes: M-cells exhibit a taut and convex plasma membrane that progressively protrudes into P-cells, which exhibit a more slack, wavy plasma membrane. These asymmetries are relaxed upon plasma membrane fusion, with observations of ramified pores that may result from multiple initiations or inhomogeneous expansion. We show that P-cells have a higher exo- to endocytosis ratio than M-cells, and that local reduction in exocytosis abrogates membrane waviness and compromises cell fusion significantly more in P- than M-cells. Reciprocally, reduction of turgor pressure specifically in M-cells prevents their protrusions into P-cells and delays cell fusion. Thus, asymmetric membrane conformations, which result from differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell-cell fusion.

scholarly journals Prm1p, a Pheromone-Regulated Multispanning Membrane Protein, Facilitates Plasma Membrane Fusion during Yeast Mating

2000 ◽  
Vol 151 (3) ◽  
pp. 719-730 ◽  
Author(s):  
Maxwell G. Heiman ◽  
Peter Walter
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Large Fraction ◽  
Cell Contact ◽  
Yeast Mating

Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell–cell contact where fusion occurs. In matings between Δprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of ∼8 nm. Thus, the phenotype of Δprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused. This phenotype suggests a role for Prm1p in plasma membrane fusion.

scholarly journals Prm1 Prevents Contact-Dependent Lysis of Yeast Mating Pairs

2004 ◽  
Vol 3 (6) ◽  
pp. 1664-1673 ◽  
Author(s):  
Hui Jin ◽  
Candice Carlile ◽  
Scott Nolan ◽  
Eric Grote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Yeast Cells ◽  
Osmotic Gradient ◽  
Fusion Event ◽  
Mating Partner ◽  
Yeast Mating ◽  
Cell Wall Remodeling ◽  
A Cell

ABSTRACT Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 μm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.

scholarly journals Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Olivia Muriel ◽  
Laetitia Michon ◽  
Wanda Kukulski ◽  
Sophie G. Martin
Keyword(s):  
Plasma Membrane ◽  
Cell Fusion ◽  
Turgor Pressure ◽  
Light And Electron Microscopy ◽  
Asymmetric Membrane ◽  
Local Reduction ◽  
Fusion Site ◽  
Actin Structure ◽  
Fusion Pores ◽  
Cell Cell

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.

scholarly journals Genetic Control of Fusion Pore Expansion in the Epidermis ofCaenorhabditis elegans

2007 ◽  
Vol 18 (4) ◽  
pp. 1153-1166 ◽  
Author(s):  
Tamar Gattegno ◽  
Aditya Mittal ◽  
Clari Valansi ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Fusion Pore ◽  
Membrane Domains ◽  
Cell Pair ◽  
C Elegans ◽  
Ultrastructural Studies ◽  
Different Temperatures ◽  
Multiple Cell ◽  
Extensive Information

Developmental cell fusion is found in germlines, muscles, bones, placentae, and stem cells. In Caenorhabditis elegans 300 somatic cells fuse during development. Although there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: the left and right membrane domains are fusion-incompetent, whereas the anterior and posterior membrane domains fuse with autonomous kinetics in embryos. All but one cell pair can initiate the formation of the largest syncytium. The first cell fusion does not trigger a wave of orderly fusions in either direction. Ultrastructural studies show that epidermal syncytiogenesis require eff-1 activities to initiate and expand membrane merger.

scholarly journals Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.

1994 ◽  
Vol 127 (6) ◽  
pp. 1885-1894 ◽  
Author(s):  
J Zimmerberg ◽  
R Blumenthal ◽  
D P Sarkar ◽  
M Curran ◽  
S J Morris
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Pore Formation ◽  
Fusion Pore ◽  
Restricted Movement ◽  
Influenza Hemagglutinin ◽  
Simultaneous Measurements ◽  
Cell Membrane Capacitance ◽  
Total Fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

scholarly journals Imaging the recruitment and loss of proteins and lipids at single sites of calcium-triggered exocytosis

2016 ◽  
Vol 27 (15) ◽  
pp. 2423-2434 ◽  
Author(s):  
Adam J. Trexler ◽  
Kem A. Sochacki ◽  
Justin W. Taraska
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Membrane Fusion ◽  
Beta Cells ◽  
Total Internal Reflection ◽  
Living Cells ◽  
Dense Core ◽  
Fusion Pore ◽  
Dense Core Vesicles ◽  
Fusion Site

How and when the dozens of molecules that control exocytosis assemble in living cells to regulate the fusion of a vesicle with the plasma membrane is unknown. Here we image with two-color total internal reflection fluorescence microscopy the local changes of 27 proteins at single dense-core vesicles undergoing calcium-triggered fusion. We identify two broad dynamic behaviors of exocytic molecules. First, proteins enriched at exocytic sites are associated with DCVs long before exocytosis, and near the time of membrane fusion, they diffuse away. These proteins include Rab3 and Rab27, rabphilin3a, munc18a, tomosyn, and CAPS. Second, we observe a group of classical endocytic proteins and lipids, including dynamins, amphiphysin, syndapin, endophilin, and PIP2, which are rapidly and transiently recruited to the exocytic site near the time of membrane fusion. Dynamin mutants unable to bind amphiphysin were not recruited, indicating that amphiphysin is involved in localizing dynamin to the fusion site. Expression of mutant dynamins and knockdown of endogenous dynamin altered the rate of cargo release from single vesicles. Our data reveal the dynamics of many key proteins involved in exocytosis and identify a rapidly recruited dynamin/PIP2/BAR assembly that regulates the exocytic fusion pore of dense-core vesicles in cultured endocrine beta cells.

scholarly journals The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

2019 ◽  
Author(s):  
Zhenyong Wu ◽  
Nadiv Dharan ◽  
Sathish Thiyagarajan ◽  
Ben O’Shaughnessy ◽  
Erdem Karatekin
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Pore ◽  
Calcium Sensor ◽  
Fusion Reactions ◽  
Synaptotagmin 1 ◽  
Pore Opening ◽  
Pore Dilation ◽  
Fusion Pores

ABSTRACTAll membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Our results suggest that fusion pore dilation by Syt1 requires interactions with SNAREs, PI(4,5)P2, and calcium. Pore opening was abolished by a mutation of the tandem C2 domain (C2AB) hydrophobic loops of Syt1, suggesting that their calcium-induced insertion into the membrane is required for pore opening. We propose that loop insertion is also required for pore expansion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. We conclude that Syt1 assumes a critical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release.SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore.

Enlarged Crystalline Patches on the Plasma Membrane of Yeast Protoplasts

1980 ◽  
Vol 38 ◽  
pp. 686-687
Author(s):  
G. Sosinsky ◽  
R. Schekman ◽  
R. Glaeser
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Plasma Membrane ◽  
High Resolution Electron Microscopy ◽  
Yeast Cells ◽  
Physiological Conditions ◽  
Resolution Electron ◽  
Yeast Protoplasts ◽  
Intramembraneous Particles ◽  
Crystalline Array

The crystalline patches of intramembraneous particles that form in the yeast plasma membrane, under stationary state physiological conditions, represent a potentially interesting specimen for high resolution electron microscopy. Isolation of these crystalline membrane patches first requires removal of the cell wall and the formation of osmotically fragile yeast protoplasts. In developing a procedure for the isolation of these crystalline membrane patches, we have found that the intramembraneous particles form much larger crystalline patches in protoplasts than in intact yeast cells. We have performed deep etch experiments and have found that the crystalline array of particles is not expressed on the extracellular surface of the plasma membrane.

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