scholarly journals Transmembrane phospholipid translocation mediated by Atg9 is involved in autophagosome formation

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Minami Orii ◽  
Takuma Tsuji ◽  
Yuta Ogasawara ◽  
Toyoshi Fujimoto
Keyword(s):  
Essential Role ◽  
De Novo ◽  
Freeze Fracture ◽  
Intensive Study ◽  
Autophagosome Formation ◽  
Symmetrical Distribution ◽  
Liposomal Membranes ◽  
Freeze Fracture Electron Microscopy ◽  
Phosphatidylinositol 4 ◽  
Fracture Electron

The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo–synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.

scholarly journals De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca2+ release

2017 ◽  
Vol 114 (52) ◽  
pp. 13822-13827 ◽  
Author(s):  
Stefano Perni ◽  
Manuela Lavorato ◽  
Kurt G. Beam
Keyword(s):  
Skeletal Muscle ◽  
Conformational Changes ◽  
De Novo ◽  
Freeze Fracture ◽  
Contraction Coupling ◽  
Skeletal Muscle Contraction ◽  
Gene Knockouts ◽  
Conformational Coupling ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of CaV1.1, located ∼12 nm away in the PM. Over the last ∼30 y, gene knockouts have revealed that CaV1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of CaV1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between CaV1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation–contraction coupling in skeletal muscle.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

scholarly journals Freeze-fracture Electron Microscopy on Microbubbles Used as Theranostics or Made of Lung Surfactant

2010 ◽  
Vol 16 (S2) ◽  
pp. 1172-1173
Author(s):  
B Papahadjopoulos-Sternberg ◽  
J Ackrell
Keyword(s):  
Electron Microscopy ◽  
Lung Surfactant ◽  
Freeze Fracture ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 223-232
Author(s):  
John F. Fallon ◽  
Robert O. Kelley
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Apical Ectodermal Ridge ◽  
Structural Requirements ◽  
Limb Morphogenesis ◽  
Vertebrate Limb ◽  
The Difference ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The fine structure of the apical ectodermal ridge of five phylogenetically divergent orders of mammals and two orders of birds was examined using transmission and freeze fracture electron microscopy. Numerous large gap junctions were found in all apical ectodermal ridges studied. This was in contrast to the dorsal and ventral limb ectoderms where gap junctions were always very small and sparsely distributed. Thus, gap junctions distinguish the inductively active apical epithelium from the adjacent dorsal and ventral ectoderms. The distribution of gap junctions in the ridge was different between birds and mammals but characteristic within the two classes. Birds, with a pseudostratified columnar apical ridge, had the heaviest concentration of gap junctions at the base of each ridge cell close to the point where contact was made with the basal lamina. Whereas mammals, with a stratified cuboidal to squamous apical ridge, had a more uniform distribution of gap junctions throughout the apical epithelium. The difference in distribution for each class may reflect structural requirements for coupling of cells in the entire ridge. We propose that all cells of the apical ridges of birds and mammals are electrotonically and/or metabolically coupled and that this may be a requirement for the integrated function of the ridge during limb morphogenesis.

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