scholarly journals Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy

1998 ◽  
Vol 95 (19) ◽  
pp. 11235-11240 ◽  
Author(s):  
S. Eskandari ◽  
E. M. Wright ◽  
M. Kreman ◽  
D. M. Starace ◽  
G. A. Zampighi
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Structural Analysis ◽  
Membrane Proteins ◽  
Freeze Fracture ◽  
Plasma Membrane Proteins ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin

2002 ◽  
Vol 282 (6) ◽  
pp. F998-F1011 ◽  
Author(s):  
Tian-Xiao Sun ◽  
Alfred Van Hoek ◽  
Yan Huang ◽  
Richard Bouley ◽  
Margaret McLaughlin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Direct Evidence ◽  
Collecting Duct ◽  
Freeze Fracture ◽  
Water Channels ◽  
Dominant Negative ◽  
Coated Pits ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated “water channels” were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK1cells expressing an AQP2- c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.

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.

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