Freeze-fracture Immunolabeling Research Articles

Different priming states of synaptic vesicles underlie distinct release probabilities at hippocampal excitatory synapses

A stunning example of synaptic diversity is the postsynaptic target cell-type-dependent difference in synaptic efficacy in cortical networks. Here, we show that CA1 pyramidal cell (PC) to fast spiking interneuron (FSIN) connections have 10-fold larger release probability (Pv) than those on oriens lacunosum-moleculare (O-LM) interneurons. Freeze-fracture immunolabeling revealed that different nano-topologies and coupling distances between Ca2+ channels and release sites (RSs) are not responsible for the distinct Pv. Although [Ca2+] transients are 40% larger in FSINs innervating boutons, when [Ca2+] entry is matched in the two bouton populations, EPSCs in O-LM cells are still 7-fold smaller. However, application of a phorbol ester analog resulted in a ∼2.5-fold larger augmentation at PC - O-LM compared to PC - FSIN synapses, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct RS occupancies and demonstrate that incompletely primed, but docked, vesicles limit the output of PC - O-LM synapses.

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae

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.

Alphavirus adsorption to mosquito cells as viewed by freeze fracture immunolabeling

Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4 h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction of the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor.

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

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.

Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton

Gangliosides have been implicated in exerting multiple physiological functions, and it is important to understand how their distribution is regulated in the cell membrane. By using freeze-fracture immunolabeling electron microscopy, we showed that GM1 and GM3 make independent clusters that are significantly reduced by cholesterol depletion. In the present study, we examined the effects of actin depolymerization/polymerization and Src-family kinase inhibition on the GM1 and GM3 clusters. Both GM1 and GM3 clustering was reduced when the actin cytoskeleton was perturbed by latrunculin A or jasplakinolide, but the decrease was less significant than that induced by cholesterol depletion. On the other hand, inhibition of Src-family kinases decreased GM3 clustering more drastically than did cholesterol depletion, whereas its effect on GM1 clustering was less significant. GM1 and GM3 were segregated from each other in unperturbed cells, but co-clustering increased significantly after actin depolymerization. Our results indicate that the GM1 and GM3 clusters in the cell membrane are regulated in different ways and that segregation of the two gangliosides depends on the intact actin cytoskeleton.

The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast

In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using freeze-fracture immunolabelling served to detect these proteins in situ. As expected from previous in planta and yeast experiments, NSP accumulated within the nuclei, whereas MP was targetted to the protoplasmic face of plasma membranes when expressed alone. Upon coexpression, NSP was localised at the plasma membranes, where it was strongly attached. These results support a model in which NSP transports viral DNA to the cell periphery to facilitate cell-to-cell movement of viral DNA within plants. In contrast to AbMV MP, no plant-specific protein seems to be necessary for the translocation of NSP to the plasma membrane.

Regulation of Lens Cell-to-Cell Communication by Activation of PKCγ and Disassembly of Cx50 Channels

Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase Cgamma (PKCgamma) affects the number of channels composed of Cx46, Cx50, or both connexins. Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKCgamma or the inactive analogue 4alpha-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKCgamma activity, connexin phosphorylation and coimmunoprecipitation. Treatment with TPA activated PKCgamma and uncoupled the lens cortex by approximately 60%. PDD had no effect. Activation of PKCgamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKCgamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKCgamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines. The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.

Spatial Integration of TIP47 and Adipophilin in Macrophage Lipid Bodies

We studied the distribution of the PAT family proteins TIP47 and adipophilin in lipid bodies of THP-1 cell-derived macrophages using freeze-fracture immunolabeling and other techniques. Lipid bodies in macrophages comprise lipid droplets and extensive, previously scantily characterized sheet-like organelles, which we descriptively call "lipid sails." TIP47 and adipophilin are components of many, but not all, the lipid droplets. Both proteins are not confined to the surface of lipid droplets, as supposed, but are also inside lipid droplet cores. They are not codistributed stoichiometrically in lipid droplets. How TIP47 and adipophilin, which are polar proteins, enter the lipid droplets and are packaged among the hydrophobic neutral lipids of the core is unclear. However, in the lipid layers of the core, these proteins are directed sometimes inward and sometimes outward. Because TIP47 and adipophilin also localize to lipid sails, lipid sails are intimately involved in intracellular lipid metabolism.

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Freeze-fracture Immunolabelling of Cytoplasmatic not Membrane-bound Proteins

Freeze-fracture Immunolabelling of Cytoplasmatic not Membrane-bound Proteins

Cholesterol transporter caveolin-1 transits the lipid bilayer during intracellular cycling.

Caveolin-1, a major protein of cell surface invaginations called caveolae, is currently believed to cycle between the plasma membrane and intracellular compartments via the endocytotic pathway, at least for part of its itinerary. We studied the distribution of caveolin-1 in cell membranes, using ultrathin cryosections and freeze-fracture immunolabeling and found this protein not only in the cytoplasmic leaflet of the plasma membrane, but also in the exoplasmic leaflet of all intracellular membranes. This sidedness implies that caveolin-1 switches from one membrane leaflet to the other somewhere on its way through the cell and rules out the classic mechanism of endocytotic membrane budding and fusion for caveolin-1 intracellular trafficking. Underlying the sidedness of caveolin-1 may be a fundamental, hitherto unrecognized, mechanism by which proteins transit membranes.