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Synaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP2 signals during induction of long-term depression (LTD). During the first minutes PIP2 rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP2 accumulation. The transiently increased PIP2 signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP2 degradation provides timely termination of PIP2 cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP2 during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP2 dynamics. Ultrahigh-resolution views of PIP2 in freeze-fractured plasma membranes of dendritic spines during induction of long-term depression reveal spatial and temporal cues set by PIP2 and reveal the molecular mechanisms behind the observed PIP2 dynamics [ABSTRACT FROM AUTHOR]

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1. [ABSTRACT FROM AUTHOR]

Many developmental processes rely on cortical actin dynamics; however, the mechanisms of its fine control at the cell cortex are still largely unknown. Our analyses demonstrate that the lipid- and F-actin-binding protein Abp1 is crucial for actin-driven bristle development in Drosophila melanogaster. Combined genetic, cell biological and biochemical analyses reveal that Abp1 triggers cortical Arp2/3-mediated actin nucleation by complex formation with Scar in bristle development. The role of the plasma-membrane-associated Abp1 subpool was highlighted by constitutively membrane-anchored Abp1. Such gain-of-function experiments led to a severe splitbristle phenotype, which was negatively correlated with bristle length. This phenotype was dependent on Scar but not on WASP and required the Scar-interacting SH3 domain of Abp1. Strikingly, knockout of abp1 led to defects in both microchaete and macrochaete bristle integrity. Importantly, Arp2- and Scar-deficient flies displayed similar bristle phenotypes. Microchaetes of flies deficient for Abp1, Arp2 and Scar functions had kinks, whereas those of wasp heterozygous flies did not. Electron microscopy analyses revealed that abp1 knockout, Arp2 RNAi and Scar RNAi all led to distorted macrochaetes with an excessive number of ridges. Interestingly, despite the physical association of Abp1 with Scar and its ability to use the Arp2/3 complex activator as an effector, abp1 knockout did not affect Scar stability. This is in contrast to classical Scar complex components, such as Kette or Sra-1. Our work reveals that Abp1 is an important, Scar-interacting factor controlling cortical Arp2/3-mediated actin nucleation and unravels a novel layer of complexity in the scrupulous control of cortical actin nucleation during sensory organ formation. [ABSTRACT FROM AUTHOR]

The freezefracture technique provides large and detailed ultrastructural views of cellular membranes in the transmission electron microscope. The major goal of freezefracture cytochemistry is the identification and localization of membrane components such as proteins or lipids for studying their distribution, dynamics or their relationship to specialized membrane domains. The most versatile and effective technique in freezefracture cytochemistry is termed freezefracture replica immunolabeling (FRIL). Using this technique chemically unfixed cellular samples are rapidly cryofixed in their natural habitat. They are fractured, replicated and immobilized by platinumcarbon evaporation followed by a careful sodium dodecyl sulfate (SDS) treatment. The detergent SDS removes most of the cellular material except those molecules that are in direct contact to the platinum/carbon replica. Immunogold labeling of these replicabound biomolecules visualizes their spatial organization in high resolution on planar views of cellular membranes. [ABSTRACT FROM AUTHOR]

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like. [ABSTRACT FROM AUTHOR]

Abstract: The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatAd was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatAd formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatAd independent from TatCd, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatAd complexes. [Copyright &y& Elsevier]

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-ß-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes. [ABSTRACT FROM AUTHOR]

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gβ). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 °C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100–400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored. [ABSTRACT FROM AUTHOR]

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases. [ABSTRACT FROM AUTHOR]