Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.     

Calcium regulation of actin dynamics in dendritic spines

Most excitatory synapses in the brain are made on spines, small protrusions from dendrites that exist in many different shapes and sizes. Spines are highly motile, a process that reflects rapid rearrangements of the actin cytoskeleton inside the spine, and can also change shape and size over longer timescales. These different forms of morphological plasticity are regulated in an activity-dependent way, involving calcium influx through glutamate receptors and voltage-gated calcium channels. Many proteins regulating the turnover of filamentous actin (F-actin) are calcium-dependent and might transduce intracellular calcium levels into spine shape changes. On the other hand, the morphology of a spine might affect the function of the synapse residing on it. In particular, the induction of synaptic plasticity is known to require large elevations in the postsynaptic calcium concentration, which depend on the ability of the spine to compartmentalize calcium. Since the actin cytoskeleton is also known to anchor postsynaptic glutamate receptors, changes in the actin polymerization state have the potential to influence synaptic function in a number of ways. Here we review the most prominent types of changes in spine morphology in hippocampal pyramidal cells with regard to their calcium-dependence and discuss their potential impact on synaptic function.     

Immunocytochemical localization of tubulin with colloidal gold in cilia of rabbit tracheal epithelial cultures

Ciliated tracheal epithelia cell cultures were investigated immunocytochemically with anti-tubulin and colloidal gold. When rabbit tracheal cultures were fixed in paraformaldehyde, treated with acetone, anti-tubulin and a second antibody coupled to FITC, fluorescence was associated with cytoskeletal and axonemal microtubules. Cilia covering the apical surface of the ciliated tracheal cells fluoresced very brightly thus facilitating identification of this cell type. Electron microscopy of tracheal cultures fixed as above, treated with Triton-X 100 and incubated in anti-tubulin and protein A coupled to colloidal gold resulted in the highly specific localization of tubulin in ciliary axonemes and basal bodies. Omission of primary or secondary antibody resulted in extremely low levels of fluorescence while no colloidal gold particles could be detected in cultures at the electron microscopy level when rabbit anti-tubulin was omitted.     

Immunocytochemical localization of actin in the nucleolus of rat oocytes

Summary— In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post-embedding ultrastructural protein-A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nucleolus.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex

An indirect immunocytofluorescence technique was used to examine the distribution of the prostaglandin-forming cyclooxygenase in the cerebellar cortex of the pig, guinea pig, rat, mouse, cow, rabbit and sheep. Cyclooxygenase antigenicity was detected (a) in the cell bodies of Bergman glial cells in the Purkinje cell layer of the porcine, ovine and bovine cerebellar cortex; (b) in small arterioles throughout the cerebellar cortex in the sheep and cow; and (c) in the endothelial cells of large arteries in all the species examined. No cyclooxygenase-positive staining was apparent in neuronal cell bodies of granule, basket, stellate or Purkinje cells. Our results establish that prostaglandin endoperoxides can be synthesized by the arterial vasculature and at least certain glial cells in the central nervous system.     

Platelet-derived growth factor labeled to colloidal gold for use as a mitogenic receptor probe

Studies by others utilizing 125I-PDGF have indicated that target cells express a high affinity surface receptor for PDGF. We have bound purified platelet-derived growth factor (PDGF) to gold colloid particles to explore the interaction of PDGF with mouse 3T3 cells. The gold-PDGF complex consists of approximately 26 PDGF molecules electrostatically absorbed to gold colloid (approximately 14.1 nm). The gold-PDGF complex induced mitogenic stimulation similar to unbound PDGF, although a 5 to 6 fold greater amount of complexed PDGF was required for the same effect. Incubation of the gold-PDGF complex with 3T3 cells for 4 h at 4 degrees C revealed that 98% of the membrane binding was randomly distributed on the cell surface with respect to coated pits, with each cell binding 7000 to 11000 complexes. Addition of a 20-fold excess of unlabeled PDGF reduced surface binding of the gold-PDGF complex by 87% (1230 probes/cell). Warming to 37 degrees C followed by time-interval fixation permitted visualization of endocytosis of the complexes in coated vesicles (1-3 min), internalization (3-15 min) and lysosomal accumulation (15-60 min). Pretreatment of cultures with monensin (2 h, 10 microM) abolished receptor binding, internalization and subsequent mitogenesis of the gold-PDGF complex. These studies support the suggestion that PDGF requires a surface receptor to elicit mitogenesis.     

Distribution of MAP 2 in dendritic spines and its colocalization with actin

Summary The distribution of MAP2 and actin in dendritic spines of the visual and cerebellar cortices, dentate fascia, and hippocampus was determined by using immunogold electron microscopy. By this approach, we have confirmed the presence of MAP2 in dendritic spines and identified substructures within the spine compartment showing MAP2 immunoreactivity. MAP2 immunolabeling was mainly associated with filaments which reacted with a monoclonal anti-actin antibody. Also, by immunogold double-labeling we colocalized MAP2 with actin on the endomembranes of the spine apparatus, smooth endoplasmic reticulum, and in the postsynaptic density. Labeling was nearly absent in axons and axonal terminals. These results indicate that MAP2 is an actin-associated protein in dendritic spines. Thus, MAP2 may organize actin filaments in the spine and endow the actin network of the spine with dynamic properties that are necessary for synaptic plasticity.     

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Using commercial monoclonal antibodies against actin and tubulin (alpha and beta), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and - most intensely - in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail. Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Summary Using commercial monoclonal antibodies against actin and tubulin ( and ), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and — most intensely — in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail.Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Ultrastructural localization of neutral DNAse in hepatocytes by immune electron microscopy using colloidal gold

A method for obtaining of the colloidal gold with particles 20 nm in diameter is described. The use of conjugate of colloidal gold-specific antibodies to the neutral DNAase is shown to determine the DNAase localization on ultrathin epontic sections of rat liver fixed by glutaraldehyde. The conditions of fixation, filling and immune reactions are described. The neutral DNAase has been found to localize mainly in heterochromatin.     

The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines

Synapse function and plasticity depend on the physical structure of dendritic spines as determined by the actin cytoskeleton. We have investigated the organization of filamentous (F-) actin within individual spines on CA1 pyramidal neurons in rat hippocampal slices. Using two-photon photoactivation of green fluorescent protein fused to beta-actin, we found that a dynamic pool of F-actin at the tip of the spine quickly treadmilled to generate an expansive force. The size of a stable F-actin pool at the base of the spine depended on spine volume. Repeated two-photon uncaging of glutamate formed a third pool of F-actin and enlarged the spine. The spine often released this "enlargement pool" into the dendritic shaft, but the pool had to be physically confined by a spine neck for the enlargement to be long-lasting. Ca2+/calmodulin-dependent protein kinase II regulated this confinement. Thus, spines have an elaborate mechanical nature that is regulated by actin fibers.     

Experience-dependent pruning of dendritic spines in visual cortex by tissue plasminogen activator

Sensory experience physically rewires the brain in early postnatal life through unknown processes. Here, we identify a robust anatomical consequence of monocular deprivation (MD) in layer II/III of visual cortex that corresponds to the rapid, functional loss of responsiveness preceding any changes in axonal input. Protrusions on pyramidal cell apical dendrites increased steadily after eye opening, but were transiently lost through competitive mechanisms after brief MD only during the physiological critical period. Proteolysis by tissue-type plasminogen activator (tPA) conversely declined with age and increased with MD only in young mice. Targeted disruption of tPA release or its upstream regulation by glutamic acid decarboxylase (GAD65) prevented MD-induced spine loss that was pharmacologically rescued concomitant with critical period plasticity. An extracellular mechanism for structural remodeling that is limited to the binocular zone upon proper detection of competing inputs thus links early sensory experience to visual function.     

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

Summary The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microseopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5–10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthognal texture of bundles of microfibrils that are thought to contain chitin. The pesence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microscopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5-10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthogonal texture of bundles of microfibrils that are thought to contain chitin. The presence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

In situ localization of antigens of Histoplasma capsulatum using colloidal gold immune electron microscopy

Histoplasma capsulatum contains multiple antigens, among them the H antigen and M antigen, which are useful in serologic testing for histoplasmosis. We prepared 7 mouse monoclonal antibodies (5 IgG, 2 IgM) to histoplasmin, and compared these with polyclonal histoplasmin antibodies raised in rabbits and mice. Both monoclonal and polyclonal antibodies were high titered by ELISA. Colloidal gold immune electron microscopy (CGIEM) showed that polyclonal antibodies to histoplasmin or H antigen bound at multiple sites in the cell wall, cytoplasm, and nucleus of Histoplasma yeast cells. In contrast, antibodies to M antigen selectively label the cell membrane and antibodies to alkali soluble cell wall antigen label only the cell wall. Polyclonal antibodies cross reacted extensively with other fungi, both by ELISA and CGIEM. Monoclonal antibodies stained only cytoplasmic epitopes, but also cross reacted with other fungi by electron microscopy. Only periodate treated H antigen elicited polyclonal antibodies which were more specific than those of untreated H antigen or histoplasmin.