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Synaptic Vesicle Isolation and Characterization, Optimization of Receptor Structure, and Structure of the Viral Membrane

Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics
Paul S. Blank, PhD, Staff Scientist
Svetlana Glushakova, PhD, Staff Scientist
Vadim A. Frolov, PhD, Senior Research Fellow
Vladimir A. Lizunov, MS, Research Fellow
Petr Chlanda, PhD, Visiting Fellow
Alexander Chanturiya, PhD, Guest Researcher
Glen Humphrey, PhD, Guest Researcher
Ludmila Bezrukov, MS, Chemist
Jane E. Farrington, MS, Contractor
Hang Waters, MS, Biologist
Mariam Ghochani, MS, Graduate Student
Alex Steinkamp, BA, Postbaccalaureate Intramural Research Training Award Fellow

We study membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells in order to understand viral and parasite infection, exocytosis, apoptosis, the mechanism of immune protection by stem cells and their cytotoxic potential, and immune dysfunction in microgravity. The overall goals of the exocytosis projects are to understand the mechanisms of cellular secretion at the physical, biophysical, and chemical levels. This process of protein secretion is the climax of the secretory pathway and operates in both constitutive and triggered ways. The process of endocytosis is equally important to retrieve membrane components.

This year, we focused on a purified fraction of exocytotic vesicles, synaptic vesicles. They 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. We developed an improved method for isolating highly pure and intact squid synaptic vesicles.

In collaboration with Sergey Bezrukov, we discovered that, at lower substrate concentrations, stronger substrate binding is required and that the deviations from optimal interaction become more critical as the substrate concentration increases, i.e., higher concentrations require more precise tuning. Thus, uniporters designed to transport the same molecule in the same cell have to be optimized with different amino-acid sequences, with one gene coding for a uniporter protein that functions most efficiently at high solute concentrations, whereas another gene is coding for one that is most efficient at low concentrations.

Negative staining can provide detailed, two-dimensional images of biological structures while combining tomography with negative staining can provide three-dimensional images. Basic requirements for a negative stain for tomography are that the density and atomic number of the stain are optimal and that the stain is not degraded by the intense electron beam needed to collect a full set of tomographic images. A commercially available, tungsten-based stain, methylamine tungstate, appears to satisfy these prerequisites. Tomograms derived from multiple projections of EM images of the same structure yielded detailed images of single proteins on the surface of influenza A virus. By comparing these images with published results from other methods, we could evaluate the negative-stain tomography. Images of surface renderings of the virus are a good fit to images derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Thus, negative stain tomography provides realistic and detailed images of individual molecules in their normal setting on the surface of influenza A virus.

Characterization and isolation of squid synaptic vesicles

Neurotransmitter release by fusion of synaptic vesicles (SV) with the pre-synaptic plasma membrane upon transient increases in intracellular Ca²⁺ is essential for propagating action potentials between neurons. SV fusion requires cooperative interactions between the lipids and proteins of both the pre-synaptic and SV membranes. Although the structures of many SV proteins have been solved, and a prototypic structural model of an individual SV has been presented, an overall picture of how proteins are organized at the vesicle surface is still lacking.

It is well established that the vertebrate and invertebrate nervous systems exhibit many similarities in terms of neuronal function. The squid (Logilo pealei) nervous system, in particular, has been used to demonstrate the neuronal resting potential, record electrical action potentials, and define the role of calcium in synaptic transmission. The squid optic lobe contains 50–80% of the neurons in the squid central nervous system and is therefore an excellent source of synaptic vesicles for the study of their biophysical and structural properties. Dowdall and Whittaker described the isolation of synaptic vesicle–rich fractions from squid optic lobe obtained by osmotic shock. However, the purity of their final fraction was never critically evaluated either by biochemical or electron microscopy techniques (Dowdall and Whittaker, 1973). Chin and Goldman used the same method to purify synaptic vesicles from frozen squid optic lobe and added controlled-pore glass chromatography as a final purification step. Based on their detailed biochemical analysis, the vesicle fraction was approximately 60% pure.

Using advances in the purification of synaptic vesicles from rat brain (Huttner et al., 1983), we optimized a SV isolation protocol for squid optic lobe to obtain a highly pure and intact SV population for biochemical and ultrastructural studies. The key step was glycerol density gradient centrifugation. Various 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. A BLAST search of a library of squid expressed-sequence tags identified 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrated directly that vesicles flatten during the drying step of negative staining and, furthermore, showed details of individual vesicles and other proteins at the vesicle surface.

To determine their purity and size distribution and the effects of different EM specimen preparation techniques on the average SV size, we evaluated the SV-enriched fractions by electron microscopy (EM). The distribution of SV size in SV-enriched fractions suggests that the SV we isolated are more than 95% pure. We used the purified vesicles to characterize the organization of the surface of individual SVs by tungstate-based negative-stain EM tomography.

Our results clearly demonstrate that estimates of SV size are dependent on the method of preparation of the SV sample for EM. Section thickness is unlikely a source of variation because we only measured vesicles for which the delimiting edges of the membrane were visible in a single section and then computed the size based on ferrets diameter with a circularity value close to unity. Fixation and processing conditions can alter the absolute dimensions of organelles; glutamatergic vesicle diameter in the rapidly frozen, freeze-substituted anteroventrocochlear nucleus is notably greater (7 nm) than those in the aldehyde-fixed hippocampus. Negatively stained, non-fixed SV samples had the largest mean diameter compared with samples obtained by other preparation techniques. It should be kept in mind that the negative-stain image is a projection of the whole vesicle while sections are often only part of a vesicle, not necessarily an equatorial section. It is also known that, when protein-containing lipid vesicles are negatively stained, the vesicles dry down and collapse, approaching the diameter of two discs with the same area, one on top, one below. Changes in vesicle shape from spheres to disk by negative staining would explain the significantly larger diameter of negatively stained SVs that we observed.

Indeed, such flattening is directly demonstrated by tomographic reconstructions. With this method, SV membranes appear to be uniformly coated with large knobs and smaller hairs. Ostensibly the knobs are protruding V-ATPase molecules, which are much larger than other SV proteins. Given that the stain does not penetrate the vesicle, its membrane is not enclosed on both sides, and it is poorly outlined by negative stain in XZ projections of tomograms, the membrane appears only as a faint boundary between stain and non-stain. Owing to the hair-like structures, the upper and lower boundaries of vesicles are hard to see except where the boundary is decorated by knobs. At that point, the knobs clearly delineate the position of the membrane. Thus, knobs on vesicle membranes near the centers of vesicles appear to rest on a flat surface suspended across the ends of the vesicles, as if on a drum rather than perched on a dome. Thus, the vesicles are flattened and a little thicker at their edges where their membranes fold back on themselves.

It remains to be determined how detergents used for vesicle isolation in previously published methods and the specificity of metal-protein surface interactions implicit to the negative-staining procedure affect the organization of proteins on the SV surface. Using negative stain–based electron microscopy, which permits imaging free from the assumptions of symmetry, classification, and averaging, to obtain tomograms of single intact synaptic vesicles isolated without detergents, we were able to make three-dimensional molecular reconstructions. While further research is needed to determine whether proteins of a specific type may cluster in restricted domains, below the structures noted above, negative-staining tomography shows a continuous sheet of protein blanketing the surfaces of synaptic vesicles.

The deeper our knowledge of both macromolecular crystal structures and the biochemical regulation of macromolecular structure in synapses grow, the clearer is our need for detailed structural analysis of synaptic membranes. Analysis of the spatial organization of these membranes helps us understand the assembly of synaptic organelles and the interactions between them, crucial to understanding how these change in time with activity and plasticity and how they traffic.

Optimization of receptor structure

Quantitative analysis of carrier parameters demonstrates that, with decreasing substrate concentration, the optimal strength of substrate-carrier interaction, which maximizes flux across the membrane, increases and requires less fine-tuning than at higher concentrations of the substrate. Many of the nutrients a cell needs for its functioning, such as sugars, amino acids, nucleotides, or organic bases, require specialized transporters to cross the cell membrane.

The rapid growth of available information, characterized as transporter explosion by Uhl and Hartig, has led to creation of a transporter classification system, with division of all transporters into channels and carriers. Channel proteins are mainly considered to function as selective pores that do not need conformational rearrangements at each substrate translocation event. We focused on optimization of carrier-facilitated transport. A carrier transfers substrates via a mechanism that includes at least four steps: (i) binding of the substrate to the carrier on one side of the membrane; (ii), carrier conformational change leading to substrate transition to the other side; (iii) dissociation of the substrate; and (iv) return of the carrier to its initial conformation/position in the membrane.

The human genome harbors 43 distinct families of transport systems that comprise over 300 isoforms of individual solute carriers. Although the majority of these transport systems are responsible for uptake of specific substrates, a substantial number of transporters are used for uptake of the same solute and often exhibit overlapping expression of multiple isoforms that exist in the same cell type. So, the question naturally arises as to why there are so many transporter isoforms. We found a possible answer by analyzing carrier-facilitated transport with the focus on the optimal efficiency of the transporter. We derived analytical expressions for the optimal values of the dissociation rate constant and the ratio of the forward and backward rates of the carrier conformational transitions, which maximize the flux.

In collaboration with Sergey Bezrukov's Section, we demonstrate that, at lower substrate concentrations, stronger substrate binding is required and that the deviations from optimal interaction become more critical as the substrate concentration increases, i.e., higher concentrations necessitate more precise tuning. Thus, uniporters designed to transport the same molecule in the same cell have to be optimized with different amino-acid sequences, with one gene encoding a uniporter protein that functions most efficiently at high solute concentrations while a different gene encodes a uniporter that is most efficient at low concentrations. Although quantitative analysis of optimization of carrier-facilitated transport was conducted almost 30 years ago for the liquid membranes of extraction technology, to the best of our knowledge it has never been applied to biological carriers.

The existence of multiple transporter isoforms that carry the same molecule is well documented for almost any important substrate. Although this variety of isoforms may seem redundant and, in principle, could be explained by the lack of strong evolutionary pressures to decrease the size of the genome, our analysis offers a different interpretation. We demonstrated that transporter efficiency is fine-tuned to specific ranges of substrate concentration so that different isoforms might be tailored to adjust their amino-acid composition for the optimal strength of substrate/transporter interactions and the transition rates between different conformations.

Structure of the viral membrane

Negative staining is a pivotal technique for visualizing the averaged structure of protein molecules, counting among its successes uncovering the organization of acetylcholine receptors and structural proteins in ribosomes, the structure and flexibility of myosin molecules, and changes in the organization of influenza hemagglutinin with pH. Averaged images of negatively stained CaMKII has provided images at a molecular level comparable to those from cryomicroscopy. Averaged images of negatively stained bacterial flagellar filaments show individual alpha-helices.

Detailed structure has been less easy to come by when negative staining has been applied to complex structures not amenable to averaging such as viruses. In particular, overlap of small components of complex structures, piled layer upon layer, obscures their structure. However, it has been shown that application of single-axis tomography to paramyxovirus negatively stained with phosphotungstic acid reveals individual spikes on the surface of the virus. The stain has been used to make three-dimensional reconstructions of single synthase molecules.

Application of tomography to negatively stained material encounters the limitation that the large electron doses needed to collect a dual-axis tomographic series can degrade the negative stain, inducing aggregation. Furthermore, heavy-metal stains, when thick enough to envelop larger structures, can form layers so dense they compromise visualization of the finest specimen details. We found that methylamine tungstate (NanoW) has an electron-scattering cross section and resistance to electron irradiation that make it suitable for dual-axis tomography of influenza A virus.

The present findings open the way to using negative-stain tomography to realize fine-molecular detail in individual viral spikes imaged near focus. While breaks in the viral envelop incident to negative stains do not appear to affect the distributions or structure of individual spikes, negative staining itself can distort proteins. Indeed, the structure of influenza A virus depends on the species of negative stain used, raising the issue of how real the structures revealed by negative-stain tomography are. For instance, influenza A virus manifests much less pleiomorphism when negatively stained with uranyl acetate than with phosphotungstic acid.

The numbers and distribution of spikes on the surface of the virus obtained by negative staining can be compared with results with other techniques. Cryomicroscopy of influenza virus, which is expected to present a realistic picture of the numbers and distribution of spikes, provides sufficient detail after averaging to compare with the distributions and sizes of the two types of spikes seen in negative-stain tomography. The ratio of the small type to the large type of spike in samples from the equator of the negatively stained virus is eight to one. By comparison, counts of spikes on two influenza A viruses viewed by cryomicroscopy yielded ratios of eight to one for one virus and six to one for the other.

Thus, dual-axis tomography of influenza A virus negatively stained with methylamine tungstate showed that the structure of individual spikes on the surfaces of the virus are consistent with their distribution and atomic structure from cryomicroscopy and X-ray crystallography. Negative stain tomography thus opens the door to examining conformations of individual protein molecules and provides a useful complementary tool for elucidating structures.

Additional Funding

Jain Foundation
Intramural AIDS Targeted Antiviral Program (IATAP)
Center for Neuroscience & Regenerative Medicine (CNRM)

Publications

Stenkula KG, Lizunov VA, Cushman SW, Zimmerberg J. Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal. Cell Metab 2010;12:250-259.
Glushakova S, Humphrey G, Leikina E, Balaban A, Miller J, Zimmerberg J. New stages in the program of malaria parasite egress imaged in normal and sickle erythrocytes. Curr Biol 2010;20:1117-1121.
Pekkurnaz G, Fera A, Zimmerberg-Helms J, Degiorgis JA, Bezrukov L, Blank PS, Mazar J, Reese TS, Zimmerberg J. Isolation and ultrastructural characterization of squid synaptic vesicles. Biol Bull 2011;220:89-96.
Zimmerberg J, Hess ST. Elongated membrane zones boost interactions of diffusing proteins. Cell 2011;146:501-503.
Berezhkovskii AM, Lizunov VA, Zimmerberg J, Bezrukov SM. Functional role for transporter isoforms in optimizing membrane transport. Biophys J 2011;101:L14-16.

Collaborators

Samuel W. Cushman, PhD, Diabetes Branch, NIDDK, Bethesda, MD
Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Bethesda, MD
Samuel T. Hess, PhD, University of Maine, Orono, ME
Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD

Contact

For more information, email zimmerbj@mail.nih.gov.

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