Direct visualization of CaMKII at postsynaptic densities by electron microscopy tomography.

Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a major component of postsynaptic densities (PSDs) involved in synaptic regulation. It has been previously shown that upon activity CaMKII from the spine reversibly aggregates at the cytoplasmic surfaces of PSDs, where it encounters various targets for phosphorylation. Targets for CaMKII are also present within the PSD, but there has been no reliable method to pinpoint whether, or where, CaMKII is located inside the PSD. Here we show that CaMKII can be mapped molecule-by-molecule within isolated PSDs using negative stain electron microscopy tomography. CaMKII molecules found in the core of the PSD may represent a pool distinct from the CaMKII residing at the cytoplasmic surface.

A negative stain for electron microscopic tomography.

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (~106 e/nm²) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

Isolation and ultrastructural characterization of squid synaptic vesicles.

Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.

Two-stage surface freezing of a single top layer in a smectic-A membrane.

The crystallization of a single liquid top layer of smectic membranes of the compound 4O.8 has been studied with grazing-incidence x-ray diffraction. As this process takes place in two steps, involving an intermediate hexatic smectic-B layer before the final crystalline-B surface structure is reached, it provides a model for melting in two dimensions. The positional order has been investigated quantitatively by measuring the scattering profiles and the associated correlation lengths. The surface liquid-hexatic phase transition is found to be continuous, while the hexatic-crystal transition is weakly first order with an abrupt change of the in-plane positional correlations. The surface phase transitions do not modify the liquid in-plane structure of the interior layers.

Smectic membranes in motion: approaching the fast limits of x-ray photon correlation spectroscopy.

The dynamics of the layer-displacement fluctuations in smectic membranes have been studied by x-ray photon correlation spectroscopy (XPCS). We report transitions from an oscillatory damping regime to simple exponential decay of the fluctuations, both as a function of membrane thickness and upon changing from specular to off-specular scattering. This behavior is in agreement with recent theories. Employing avalanche photodiode detectors and the uniform filling mode of the synchrotron storage ring, the fast limits of XPCS have been explored down to 50 ns.