Kinesin associates with anterogradely transported membranous organelles in vivo

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.     

Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo

Brain dynein is a microtubule-activated ATPase considered to be a candidate to function as a molecular motor to transport membranous organelles retrogradely in the axon. To determine whether brain dynein really binds to retrogradely transported organelles in vivo and how it is transported to the nerve terminals, we studied the localization of brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using affinity-purified anti-brain dynein antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Interestingly, brain dynein accumulated both at the regions proximal and distal to the ligation sites and localized not only on retrogradely transported membranous organelles but also on anterogradely transported ones. This is the first evidence to show that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activates brain dynein only for retrograde transport.     

The transport of organelles in axons

The fast anterograde and retrograde transport systems in axons convey organelles from the soma, where synthesis occurs, to the synaptic region and back. Studies of label incorporation into newly synthesized organelles show that they move along the axon with profiles resembling traveling waves. The underlying mechanism appears to be that cross-bridging “engines” attach to the organelles and this complex then attaches by the engines to the surface of microtubules, resulting in translocation of the organelles. A model incorporating this mechanism predicts traveling-wave-like profiles of labeled organelles and can serve to link mechanistic information with fast transport measurements in intact axons. Analysis of the simplest case provides insight into the factors determining the speed and shape of the wavelike profile.     

Relationships between membranous organelles in amoebae studied by electron microscopic cytochemical staining

Summary The intracellular location of a variety of enzymes was studied in Amoeba proteus with the use of electron microscopic cytochemical methods, in an attempt to assess the relationships between different membranous organelles. One group of enzymes, including nucleoside diphosphatases (IDPase, UDPase, GDPase, ADPase), carbamoyl phosphatase, alkaline phosphatase, and BAXD oxidase was localized mainly in the rough endoplasmic reticulum, nuclear envelope, and convex side of the Golgi apparatus. Esterase activity had a similar localization except that the Golgi apparatus was "stained" throughout most of its extent. A second group of enzymes was found in Golgi cisternae and vesicles, and in some vacuoles. This group included acid phosphatase, thiamine pyrophosphatase, and aryl sulfatase. Some enzymes previously detected in cytoplasmic membranes of other cells, including glucose-6-phosphatase, showed little or no activity in amoebae. The results suggest that there are chemical similarities and probable functional relationships between the rough endoplasmic reticulum, the nuclear envelope, and the convex side of the Golgi apparatus. On the other hand, the concave pole of the Golgi apparatus, aggregates of smooth tubules and vesicles, and the cell surface appear more closely related to one another than to the endoplasmic reticulum and the convex side of the Golgi apparatus. The cytochemical similarity between the Golgi apparatus and certain vacuoles such as food vacuoles may reflect the role of the Golgi apparatus in the formation of lysosomes. The locations of reaction products of the various enzymes in amoebae are compared with observations reported for other cell types.Supported by a research grant (VC-169) from the American Cancer SocietyThe author is indebted for technical assistance to Mrs. Sue Thompson and Mrs. Christine Folsom-Kovarik     

Electron microscopic identification of the yeast spliceosome.

We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.     

Altered spectrum of retrogradely transported axonal proteins in p-bromophenylacetylurea neuropathy

The composition of retrogradely transported axonal proteins was examined by acrylamide gel electrophoresis and gel autoradiography in the experimental neuropathy induced in rats by p-bromophenylacetylurea (BPAU). Protein composition was normal during the early phase of retrograde transport but showed significant abnormalities during a later phase. The early phase consisted of proteins collected distal to a mid-thigh ligature of sciatic nerve between 15 and 24 hours after injection of [³⁵S] methionine into lumbar ventral horn of the spinal cord. In terms of their relative labeling and electrophoretic mobility, these proteins were almost identical in experimental and control rats. Most of the labeled protein bands were also identical in the later phase, collected between 24 and 48 hours, but there were some consistent omissions and additions. Present in controls but missing in BPAU treated rats were three bands at 42, 41, and 25 KDa. In contrast, 4 bands (63, 56, 50, 26 KDa) were more prominent in the experimental rats than in controls. We suspect abnormal post-translational modification or proteolysis of rapidly transported proteins in the terminal or preterminal portion of the neurons exposed to BPAU. This abnormality, in addition to a previously reported premature processing of transported organelles, may underlie the development of peripheral neuropathy.     

Electron microscopic investigation of adrenergic and non-adrenergic axons in the rabbit SA-node

Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO₄ which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.This work was supported by grants from Åhlén-Stiftelsen, the Faculty of Medicine, University of Lund, Lund, Sweden, the United States Public Health Service (project 06701-04) and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05).     

Peroxisomes exist in growth cones and move anterogradely and retrogradely in neurites of PC12D cells

Localization and movement of peroxisomes have been investigated in neurites of a subline of PC12 pheochromocytoma cells (PC12D cells). The cells were transfected with a construct encoding the green fluorescent protein and bearing the C-terminal peroxisomal targeting signal 1 SKL motif (-Ser-Lys-Leu-COOH). Peroxisomes were detected as green punctate fluorescent signals. Many peroxisomes were observed in neurites of PC12D cells, especially in neural terminal-like structures, growth cones, varicosities, and branch points. Growth cones containing many peroxisomes were active, since they extended several long filopodias. Existence of peroxisomes in growth cones and neuronal terminal-like structures suggests that peroxisomes might have some role in neuronal extension and nerve terminal functioning. Peroxisomal motility was analyzed by time-lapse imaging using a fluorescence microscope at 25 degrees C. Peroxisomes were transported bidirectionally in neurites, i.e., through anterograde and retrograde transport. This result suggests that peroxisomes move to growth cones and neural terminals from the PC12D cell body, play some role in these parts, and go back to cell body.     

Electron microscopic localization and experimental modification of NADPH-diaphorase activity in crustacean sensory axons

The origin and ultrastructural localization of NADPH-diaphorase (NADPH-d) in the olfactory afferent pathway of the crayfishPacifastacus leniusculus was investigated by means of histochemical techniques. Sensory axons in the antennular nerve and the olfactory lobe glomeruli of normal animals expressed NADPH-d staining properties. The NADPH-d staining of each glomerulus was regionalized showing pronounced staining in the apical cap-region. Following ablation of the chemosensory input for 30 days, the staining properties of the antennular nerve and the glomeruli were reduced. At the electron microscopic level, the NADPH-d precipitate was found to be distributed on various membranes in neuronal profiles and glial cells. Stained neuronal profiles were frequently observed in the glomeruli, whereas the number of positive glial cells was low. Almost all NADPH-d positive profiles in the neuropil had an intraglomerular localization. The present findings suggest that NADPH-d in the crayfish olfactory lobe neuropil is localized to terminals of olfactory sensory axons.     

Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.     

Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method

The elaborate cross-connections among membranous organelles (MO), microtubules (MT), and neurofilaments (NF) were demonstrated in unifixed axons by the quick-freeze, deep-etch, and rotary-shadowing method. They were categorized into three groups: NF-associated cross-linker, MT-associated cross-bridges, and long cross-links in the subaxolemmal space. Other methods were also employed to make sure that the observed cross-connections in the unfixed axons were not a result of artifactual condensation or precipitation of soluble components or salt during deep-etching. Axolemma were permeablized either chemically (0.1% saponin) or physically (gentle homogenization), to allow egress of their soluble components from the axon; or else the axons were washed with distilled water after fixation. After physical rupture of the axolemma or saponin treatment, most of the MO remained intact. MT were stabilized by adding taxol in the incubation medium. Axons prepared by these methods contained many longitudinally oriented NF connected to each other by numerous fine cross-linkers (4-6 nm in diameter, 20-50 nm in length). Two specialized regions were apparent within the axons: one composed of fascicles of MT linked with each other by fine cross-bridges; the other was in the subaxolemmal space and consisted of actinlike filaments and a network of long cross-links (50-150 nm) which connected axolemma and actinlike filaments with NF and MT. F-actin was localized to the subaxolemmal space by the nitrobenzooxadiazol phallacidin method. MO were located mainly in these two specialized regions and were intimately associated with MT via fine short (10-20 nm in length) cross-bridges. Cross-links from NF to MO and MT were also common. All these cross-connections were observed after chemical extraction or physical rupture of the axon; however, these procedures removed granular materials which were attached to the filaments in the fresh unextracted axons. The cross-connections were also found in the axons washed with distilled water after fixation. I conclude that the cross- connections are real structures while the granular material is composed of soluble material, probably protein in nature.     

Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion

In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.     

Simultaneous ultrastructural demonstration of retrogradely transported horseradish peroxidase and choline acetyltransferase immunoreactivity

Summary In order to study the synaptic connections of neurons identified by their projection target and neurotransmitter content, we have adapted a method of combining retrograde tracing of horseradish peroxidase (HRP) and immunocytochemistry at the electron microscopic level. HRP was injected into the rat amygdala. Sections from the rostral forebrain were processed according to the 3,3-diaminobenzidine/glucose oxidase reaction followed by choline acetyltransferase (ChAT) localization. Neurons in the ventral pallidum which contained both the diffuse immunoperoxidase reaction product (ChAT) and large electron dense bodies characteristic of retrogradely transported HRP were defined as double labeled, i.e. cholinergic neurons that project to the amygdaloid body.     

Ultrastructural visualization of anterogradely transported horseradish peroxidase in developing corticospinal tract of rat

Until now a satisfactory method for electron microscopic (EM) detection of anterogradely transported horseradish peroxidase (HRP) in developing neural tissue, using sensitive chromogen tetramethylbenzidine (TMB), has not been described. Use of the stabilizing agent ammoniumheptamolybdate (AHM), in combination with a modified prolonged osmication [4 hr at pH 5.0 in 0.1 M phosphate buffer (PB)] made possible visualization of HRP-TMB-(AHM) reaction product at the ultrastructural level in outgrowing corticospinal tract (CST) fibers of young postnatal rat. This reaction product appeared to be very distinctive and clearly detectable, making ultrastructural identification of HRP-labeled outgrowing CST fibers in rat spinal cord rather easy. In addition, the procedure described in this report preserves the ultrastructural details of the developing neural tissue.     

Simultaneous ultrastructural demonstration of retrogradely transported horseradish peroxidase and choline acetyltransferase immunoreactivity

In order to study the synaptic connections of neurons identified by their projection target and neurotransmitter content, we have adapted a method of combining retrograde tracing of horseradish peroxidase (HRP) and immunocytochemistry at the electron microscopic level. HRP was injected into the rat amygdala. Sections from the rostral forebrain were processed according to the 3,3'-diaminobenzidine/glucose oxidase reaction followed by choline acetyltransferase (ChAT) localization. Neurons in the ventral pallidum which contained both the diffuse immunoperoxidase reaction product (ChAT) and large electron dense bodies characteristic of retrogradely transported HRP were defined as double labeled, i.e. cholinergic neurons that project to the amygdaloid body.