Fusion pore or porosome: structure and dynamics

Electrophysiological measurements on live secretory cells almost a decade ago suggested the presence of fusion pores at the cell plasma membrane. Membrane-bound secretory vesicles were hypothesized to dock and fuse at these sites, to release their contents. Our studies using atomic force microscopy on live exocrine and neuroendocrine cells demonstrate the presence of such plasma membrane pores, revealing their morphology and dynamics at near nm resolution and in real time.

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Fusion Pore in Live Cells

Physiology ◽

10.1152/nips.01394.2002 ◽

2002 ◽

Vol 17 (6) ◽

pp. 219-222 ◽

Cited By ~ 2

Author(s):

Bhanu P. Jena

Keyword(s):

Atomic Force Microscopy ◽

Live Cells ◽

Fusion Pore ◽

Secretory Cells ◽

Secretory Vesicles ◽

Force Microscopy ◽

Atomic Force ◽

Electrophysiological Measurements ◽

First Time ◽

Fusion Pores

Earlier electrophysiological measurements on live secretory cells suggested the presence of fusion pores at the plasma membrane, where secretory vesicles fuse to release vesicular contents. Recent studies using atomic force microscopy demonstrate for the first time the presence of the fusion pore and reveal its morphology and dynamics at near-nanometer resolution and in real time.

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Dynasore inhibits rapid endocytosis in bovine chromaffin cells

AJP Cell Physiology ◽

10.1152/ajpcell.00562.2008 ◽

2009 ◽

Vol 297 (2) ◽

pp. C397-C406 ◽

Cited By ~ 12

Author(s):

Chia-Chang Tsai ◽

Chih-Lung Lin ◽

Tzu-Lun Wang ◽

Ai-Chuan Chou ◽

Min-Yi Chou ◽

...

Keyword(s):

Plasma Membrane ◽

Chromaffin Cells ◽

Membrane Capacitance ◽

Neuroendocrine Cells ◽

Maximal Increase ◽

Force Microscopy ◽

Vesicle Recycling ◽

Atomic Force ◽

Bovine Chromaffin Cells ◽

Patch Configuration

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all ∼110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.

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Multimodal Atomic Force Imaging of Open Hemichannelinduced Modulation of Cell Volume and Viscoelastic Properties

Microscopy and Microanalysis ◽

10.1017/s1431927600018316 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 998-999

Author(s):

Seung K. Rhee ◽

Arjan P. Quist ◽

Hai Lin ◽

Nils Almqvist ◽

Ratneshx Lai

Keyword(s):

Plasma Membrane ◽

Cell Volume ◽

Lucifer Yellow ◽

Single Cells ◽

Dye Uptake ◽

Aortic Endothelial Cells ◽

Atomic Force ◽

Cell Plasma ◽

Uptake Assay ◽

Gap Junctional

Hemichannels from two single cells can join upon contact between these cells to form gap junctions - an intercellular pathway for the direct exchange of ions and small metabolites. Using techniques of fluorescent dye-uptake assay, laser confocal fluorescence imaging and atomic force microscopy (AFM), we have examined the role of hemichannels, present in the non-junctional regions of single cell plasma membrane, in the modulation of cell volume.Antibodies against a gap junctional protein connexin43, were immunolocalized to nonjunctional plasma membrane regions of single BICR-MlRk k (breast tumor epithelial) cells, KOM-1 (bovine aortic endothelial) cells, and GM04260 (AD-free human) fibroblast cells. In the absence of extracellular calcium, cytoplasmic uptake of Lucifer yellow (LY) but not of dextran-conjugated LY was observed in single cells. Dye uptake was prevented by gap junctional inhibitors, ẞ-glycyrrhetinic acid (ẞGCA) and oleamide.

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Imaging of Xenopus laevis Oocyte Plasma Membrane in Physiological-Like Conditions by Atomic Force Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927613001682 ◽

2013 ◽

Vol 19 (5) ◽

pp. 1358-1363 ◽

Cited By ~ 3

Author(s):

Massimo Santacroce ◽

Federica Daniele ◽

Andrea Cremona ◽

Diletta Scaccabarozzi ◽

Michela Castagna ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Plasma Membrane ◽

High Resolution ◽

Membrane Proteins ◽

Xenopus Laevis ◽

Functional Study ◽

Xenopus Laevis Oocyte ◽

Force Microscopy ◽

Atomic Force ◽

Afm Imaging

AbstractXenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

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Programmed ER fragmentation drives selective ER inheritance and degradation in budding yeast meiosis

10.1101/2021.02.12.430990 ◽

2021 ◽

Author(s):

George M. Otto ◽

Tia Cheunkarndee ◽

Jessica M. Leslie ◽

Gloria A. Brar

Keyword(s):

Plasma Membrane ◽

Cell Differentiation ◽

Budding Yeast ◽

Developmentally Regulated ◽

Selective Degradation ◽

Cell Plasma ◽

Membrane Tethering ◽

Membrane Bound ◽

Yeast Meiosis ◽

And Function

AbstractThe endoplasmic reticulum (ER) is a membrane-bound organelle with diverse, essential functions that rely on the maintenance of membrane shape and distribution within cells. ER structure and function are remodeled in response to changes in cellular demand, such as the presence of external stressors or the onset of cell differentiation, but mechanisms controlling ER remodeling during cell differentiation are not well understood. Here, we describe a series of developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. During meiosis, the cortical ER undergoes fragmentation before collapsing away from the plasma membrane at anaphase II. This programmed collapse depends on the meiotic transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER-plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which is regulated by the developmentally timed expression of the autophagy receptor Atg40. Autophagy relies on ER collapse, as artificially targeting ER proteins to the cortically retained ER pool prevents their degradation. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.

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3pM_K3Non-invasive Live-imaging of Plasma Membrane and Cortical Actin Dynamics in Endocytic Process by High-speed Atomic Force Microscopy

Microscopy ◽

10.1093/jmicro/dfy089 ◽

2018 ◽

Vol 67 (suppl_2) ◽

pp. i10-i10

Author(s):

Aiko Yoshida ◽

Yoshitsuna Itagaki ◽

Yuki Suzuki ◽

Shige H. Yoshimura

Keyword(s):

Atomic Force Microscopy ◽

Plasma Membrane ◽

High Speed ◽

Live Imaging ◽

Actin Dynamics ◽

Cortical Actin ◽

Force Microscopy ◽

Atomic Force

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Cell Secretion: Current Structural and Biochemical Insights

The Scientific World JOURNAL ◽

10.1100/tsw.2010.193 ◽

2010 ◽

Vol 10 ◽

pp. 2054-2069 ◽

Cited By ~ 8

Author(s):

Saurabh Trikha ◽

Elizabeth C. Lee ◽

Aleksandar M. Jeremic

Keyword(s):

Plasma Membrane ◽

Secretory Vesicles ◽

Intercellular Signaling ◽

Cell Secretion ◽

Membrane Structures ◽

Calcium Dependent ◽

Cell Plasma ◽

Membrane Bound ◽

And Control ◽

Fusion Pores

Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent) secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

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