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Correspondence to: Jorge E. Moreira, Departamento de Morfologia, Faculdade de Medicina (FMRP-USP), 14049-900 Ribeirao Preto, Sao Paulo, Brazil.
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Summary |
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The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.
(J Histochem Cytochem 46:847854, 1998)
Key Words: freeze-substitution, immunoelectron microscopy, soluble proteins, membrane proteins
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Introduction |
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CELLS GENERALLY CONTAIN soluble proteins as well as proteins attached to various organelles. The kinesin family motor proteins are no exception (
There are several reasons to doubt that current immunochemical methods give an accurate picture of the distribution of soluble or readily solubilized proteins in a living cell (
A solution to these problems might start with a physical fixation, such as rapid freezing, which would have the advantages that no fixatives would have to diffuse into the cell and that the fixation could be completed in a fraction of a msec (
A solution to this conumdrum is to use a freeze-substitution method (
We expected this method to be less sensitive than the pre-embedding methods but we also expected that much of the redistribution of proteins during processing for immunocytochemistry would be prevented. We were pleased to find that low background staining compensated for the decreased sensitivity. Although our initial interest was in the overall distribution of the kinesins, this method appeared to have wider potential applications when we tested it with several other preparations that ordinarily present difficulties with localizing soluble proteins or membrane-associated proteins present in small amounts. Here we present the results with localizing the kinesins in the squid giant axon as well as the results with other soluble and membrane-associated antigens.
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Materials and Methods |
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All tissues were first rapid-frozen with a Life Cell CF-100 freeze-slamming apparatus (Life Cell; The Woodlands, TX) (manuscript in preparation). Squid (Loligo pealii) axons were extruded onto a square of 4% agar lying on an aluminum freezing stage (a disk of aluminum 13 mm in diameter to which a circle of filter paper, 6 mm in diameter, is attached with epoxy glue) (
Kinesin was labeled with an affinity-purified rabbit IgG made to a 394 amino-acid synthetic peptide from the N terminus of the motor domain of squid kinesin heavy chain (SK-394) (-subunit was a gift from Ronald Petralia (
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Frozen samples for immunocytochemistry that were freeze-substituted on their specimen carriers were rocked at -80C in 0.1% uranyl acetate in acetone for 24 hr, warmed up to -60C, and rinsed twice in acetone (10 min each) and once in methanol. Freshly mixed K11M resin was bubbled at room temperature (RT) with dry nitrogen to mix components and to reduce contact with oxygen. Infiltration with resin diluted to 50% in acetone was followed by 75% resin and then by two changes of 100% resin at -60C, allowing 24 hr for each change. Samples were then placed in aluminum dishes, sealed with Saran plastic wrap, and polymerized with 300 nm UV for 2 days, starting at -60C, and warming up in 12-hr steps to -40C, -10C, and RT. After polymerization, the samples were placed under vacuum overnight. Cryopolymerization by UV takes almost 48 hr to complete, so there is little possibility of heating from the exothermic reaction (
Samples of axoplasm for structural comparison were prepared by substituting them at -80C with osmium tetroxide in acetone containing 4% osmium tetroxide for 36 hr (
All immunocytochemistry was performed on conventional thin plastic sections. These were cut at 7090 nm on water in a Reichert Ultracut S ultramicrotome, collected on Formvar-coated nickel grids, and immunostained with protein Agold without etching (
Quantitative analysis was performed with a Zidas digitizing system (Carl Zeiss; Thornwood, NY) interfaced with a Macintosh computer. Sets of five grids of three different axoplasm preparations were incubated with the antibodies SK 394, neurofilament, or their corresponding preimmune sera. Thirty micrographs from each conditionkinesin, neurofilament, and controlwere taken from the edge where the tissue had contacted the freezing block.
The cytosolic area was measured by subtracting the area corresponding to each organelle from the total area of the photograph in micrographs at x 50,000 final magnification. Conventions for counting were based on the dimensions of kinesin because vesicles moving along microtubules lie 1618 nm from the microtubule (
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Results |
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Axoplasm
Axoplasm prepared by conventional freeze-substitution (
Samples substituted for immunocytochemistry in the absence of osmium showed finer and more distinct cytoskeletal details than those substituted with osmium (Figure 1B). Among the various longitudinal cytoskeletal elements, the parallel microfilaments are presumed to be principally neurofilaments (
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Structure of Muscle, Cerebellum and Cultured Cells
Aplysia muscle and MDCK cells substituted without osmium and embedded in Lowicryl showed well-defined cytoplasmic structure and membrane outlining. Tight junctions on the MDCK cells and their anchoring filaments showed a clarity of structure comparable to similar structures reported previously with aldehyde fixation (Figure 5 in
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Immunoelectron Microscopy
Various combinations of aldehyde fixatives, as well as several different embedding resins, were tried in conjunction with freeze-substitution, but substantial labeling was obtained only with Lowicryl K11M low-temperature embedding after substitution without fixatives. The kinesin antibody followed by protein Agold (15 nm) provided a large amount of labeling with little nonspecific background.
The SK-394 labeling in the axoplasm showed gold particles more concentrated around organelles and microtubules, although organelles varied considerably with respect to the amount of labeling on their surfaces. The most consistently labeled organelles were vesicles, which could have up to five gold particles uniformly distributed around their perimeter. Gold particles were distributed rather than clustered at vesicle surfaces, suggesting that neither the kinesins on the vesicle surface nor the protein Agold complexes had clumped (Figure 2A and Figure 2B).
Labeling with the neurofilament antibody was performed in parallel with kinesin labeling to compare the distribution of a widely distributed and relatively stable cytoskeletal element. Neurofilament label appeared as a faintly linear arrangement of gold particles throughout the axoplasm, without any increase around organelles or microtubules (Figure 2C). The twitchin protein is another example of a soluble protein that could be localized in Aplysia muscle tissue by the freeze-substitution method. Gold particles were clearly associated with the contractile elements, often concentrated at their ends (Figure 2D).
The antibodies against membrane proteinsZO-1 and a glutamate receptorproduced precise localizations with almost no background. After incubations with anti ZO-1 protein, discrete distributions of gold particles showed a distribution at the edges of tight junctions consistent with the expected location of the ZO-1 protein (Figure 3A). The -subunit of a glutamate receptor present in cerebellar cortex synapses was marked by three to five gold particles regularly distributed along postsynaptic densities in Purkinje cell dendritic spines (Figure 3B), and therefore was also distributed exactly as expected.
Measurement of the Distribution of Kinesin Label
The localizations of ZO-1 and glutamate receptor were so focal and accompanied by so little background that they did not require morphometric analysis. The kinesins were, however, expected to be distributed throughout several cellular compartments, including the cytoplasm. The distribution of gold particles corresponding to SK-394 anti-kinesin, as well as anti-neurofilament labeling, was measured particle by particle on photographic prints comparable to those in Figure 2. Vesicles accounted for 36.4% and ER for 8.9% of the total gold count, whereas mitochondria were only 1.3% of the total (Table 1). Added together, the organelles accounted for almost half of the total cytoplasmic label. The other half was distributed between microtubules (31.4%) and the cytosol (21.9%). Because the background label over empty plastic was 0.3 particles/µm2, the contribution of background to the distribution of organelle label was negligible, although it could account for as much as 15% of the raw cytoplasmic label. Whereas half of the kinesin labeling was in cytosol and associated with microtubules, label was concentrated 57-fold around vesicles compared to cytosol, 23.5-fold around ER cisternae, 12-fold around microtubules, and fivefold around mitochondria (Table 1).
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Discussion |
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The labeling method used here combines structural definition sufficient to recognize individual cytoskeletal elements and organelles with sensitive labeling of cytoplasmic proteins. The structure of axoplasm prepared by this method is, in general, comparable to that of axoplasm substituted with osmium tetroxide. However, the finest cytoskeletal elements, which are typically obliterated or coagulated by the osmium substitution, are clearly visible in the axoplasm prepared for immunochemistry.
The accelerated substitution protocol was designed to provide minimal opportunity for soluble cytoplasmic proteins to diffuse during specimen preparation (
Another advantage of the freeze-substitution method is that it appears to enhance the sensitivity of the antibody labeling in the absence of a crosslinking fixative. Furthermore, the background labeling, to judge by the amount of label over regions lacking tissue, is very low (see Table 1), resulting in a very high signal-to-noise ratio. Thus, an antibody that recognizes many members of the kinesin family, such as AK-493, permitted the relative partitioning of various kinesins between different cellular compartments to be estimated from the distribution of gold grains.
The present results are in agreement with current biochemical studies of kinesin (
The substitution method appeared to detect kinesin attached to axoplasmic organelles as well as the soluble kinesins. The separations of gold particles clustered around organelles from the organelle surfaces were consistent with what is known about the lengths of kinesin bridges, when the size of the IgGprotein A complex is accounted for (
Another soluble protein, twitchin, that may participate in excitationcontraction coupling is abundant in muscle fibers of invertebrates, but its localization in Aplysia had proved difficult to determine by conventional immunocytochemical methods (
Different types of organelles would be expected to have different species of kinesin on them (
Proteins associated with tight junctions, such as ZO-1, have been localized with immunoelectron microscopy using pre- or postembedding procedures with only dilute paraformaldehyde as a fixative (
For proteins lying in the plane of a membrane, such as metabotropic glutamate receptors, localization has been possible using pre-embedding methods and chemical fixation (-subunit of a glutamate receptor was clearly decorated by an antibody followed by 10-nm protein Agold, showing specific postsynaptic localization. In this case, the distribution of gold particles just large enough to detect against the synaptic cleft and postsynaptic membrane material provides a precise localization of the receptor along the postsynaptic membrane at synapses of parallel fibers on Purkinje spines (
The addition of uranyl acetate with unfixed samples does not reduce labeling (
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Acknowledgments |
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We thank Dr Audrey Glauert for critical reading of the initial version of this manuscript, Mr James Tomlin for the interfacing of the Zidas digitizing system to the Macintosh computer, Dr Ayse Dosemesci for the immunoblot, Dr Harish Pant for the anti-neurofilament antibody, Dr Ronald Petralia for the incubations of the Lowicryl thin sections of cerebellum, Dr Ferdinand Vilim for the anti-twitchin antibody, and Mr John Chludzinski for expert photographic help.
Received for publication September 11, 1997; accepted March 16, 1998.
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