Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture - PubMed (original) (raw)
Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture
E R Torre et al. J Neurosci. 1996.
Abstract
There is increasing evidence that certain mRNAs are present in dendrites and can be translated there. The present study uses two strategies to evaluate whether dendrites also possess the machinery for protein glycosylation. First, precursor labeling techniques were used to conjunction with autoradiography to visualize glycosyltransferase activities that are characteristic of the rough endoplasmic reticulum (RER) (mannose) or the Golgi apparatus (GA) (galactose and fucose) in dendrites that had been separated from their cell bodies and in intact neurons treated with brefeldin A or low temperature. Second, immunocytochemical techniques were used to define the subcellular distribution of proteins that are considered markers of the RER (ribophorin I) and GA (p58, alpha-mannosidase II, galactosyltransferase, and TGN38/41). Autoradiographic analysis revealed that isolated dendrites incorporated sugar precursors in a tunicamycin-sensitive and protein synthesis-dependent manner. Moreover, when intact neurons were pulse-labeled with 3H-labeled sugars at low temperature or after treatment with brefeldin A, labeling was distributed over proximal and sometimes distal dendrites. Immunolabeling for RER markers was predominantly localized in cell bodies but extended for a considerable distance into dendrites of all neurons. Immunolabeling for GA markers was confined to the cell body in approximately 70% of the neurons, but in 30% of the neurons, the staining extended into proximal and middle dendrites. These results indicate that the machinery for glycosylation extends well into dendrites in many neurons.
Figures
Fig. 1.
Vitality of isolated dendrites. Hippocampal neurons were cultured for 10 d on a double-surface coverslip (A) as described in Materials and Methods. The Nucleopore membrane containing the cell bodies was peeled off, leaving a mesh of amputated processes on the second surface. Isolated processes were pulse-labeled with 100 μCi/ml [3H]leucine for 1 hr immediately after the isolation (0 hr) or 3, 6, and 12 hr after the isolation. The processes were fixed-stained for MAP2 and DNA and prepared for autoradiography. The graph in B shows the percentage of labeled dendrites at the different survival times. The percentage of living dendrites slowly decreases with time and drops dramatically by 6 and 12 hr after isolation. Dendrites were considered labeled at low levels when they had scattered patches of 2–3 silver grains along their length. Those processes having strings of densely packed silver grains were counted as highly labeled dendrites. The_numbers_ in parentheses represent the number of MAP2-stained processes counted in nine coverslips from three different experiments. The photographs are representative fields showing the incorporation of [3H]leucine by isolated dendrites at different times after the cut. Scale bar, 25 μm.
Fig. 2.
Mannose incorporation in isolated dendrites. Neurites isolated from 10- to 12-d-old cultures were preincubated for 1 hr in low-glucose medium, pulse-labeled with 400 μCi/ml [3H]mannose for 1 hr, rinsed in medium containing 10 m
m
cold sugar, and fixed and immunostained for MAP2.A_–_D, Sites of [3H]mannose incorporation evaluated by autoradiography and dark-field microscopy.E_–_G, MAP2 staining shows colocalization with silver grains. The arrows indicate the location of dendrites. The asterisk indicates the presence of a labeled neuron that was identified by the DNA staining. Scale bar, 25 μm.
Fig. 4.
Labeling of isolated dendrites was depressed by inhibiting the synthesis and N-glycosylation of proteins but not by competing with surface glycosyltransferases. Isolated dendrites were preincubated for 1 hr in low-glucose medium containing 50 μg/ml cycloheximide (C, H), 5 μg/ml tunicamycin (E, I), or 5 m
m
UDP–galactose (J); control cells (A, G) were incubated with no drugs. Cells were pulse-labeled with [3H]mannose (upper panel) or [3H]galactose (lower panel) for 1 hr, washed in medium containing 10 m
m
cold sugar, and fixed and immunostained for MAP2 (B, D, F; in_G_–J, immunofluorescence for MAP2 was combined with dark-field illumination). The _arrows_indicate the location of dendrites. Scale bar, 25 μm.
Fig. 5.
Effect of the inhibition of N-glycosylation, protein synthesis, and surface glycosyltransferases on the labeling of isolated dendrites with the tritiated sugar precursors mannose, galactose, and fucose. Neurites isolated as described in Materials and Methods were treated with tunicamycin (5 μg/ml), cycloheximide (50 μg/ml), or the impermeant sugar precursors UDP–galactose (5 m
m
) and pulse-labeled for 1 hr with tritiated sugars. The_bars_ represent the proportion of labeled and unlabeled MAP2-stained processes ± SEM after the different treatments. The numbers in parentheses indicate the number of dendrites evaluated in at least two different experiments. Controls were compared with the incorporation of [3H]leucine by isolated dendrites in similar conditions.
Fig. 3.
Galactose and fucose incorporation in isolated dendrites. Neurites isolated from 10- to 12-d-old cultures were preincubated for 1 hr in low-glucose medium, pulse-labeled with 200 μCi/ml [3H]fucose or 100 μCi/ml [3H]galactose for 1 hr, rinsed in medium containing 10 m
m
cold sugar, and fixed and immunostained for MAP2.A, C, Autoradiographic localization of the sites of [3H]galactose incorporation.E, G, Autoradiographic localization of [3H]fucose incorporation. B,D, F, H, The parts show that the sugar incorporation is localized over MAP2-stained processes. The arrows indicate the localization of dendrites identified by MAP2 staining. The asterisk indicates the presence of a labeled neuron that was identified by the DNA staining. Scale bar, 25 μm.
Fig. 8.
Dendritic localization of different Golgi compartments in hippocampal neurons in culture. Fourteen-day-old cultures were stained for p58 (A), α-ManII (B), β-galactosyltransferase (βGalT) (C), or TGN 38/41 (D). Stained puncta were observed concentrated within the cell body and frequently extending into dendrites that were identified by double staining for MAP2 (arrows). The staining was not found in axons (open triangles). E, Distribution of different Golgi compartments in 14- to 20-d-old hippocampal neurons in culture. Stained cells were counted and classified according to whether the immunostained structures were localized only in the cell body, in the cell body and one dendrite, or in the cell body and several dendrites. The bars represent the percentage ± SEM of cells having a given staining distribution. The_numbers_ in parentheses represent the number of cells counted for each antibody tested. The lower section shows the effect of brefeldin A on the localization of Golgi organelles. Fifteen-day-old neurons were stained for α-mannosidase II after treatment with 5 μg/ml brefeldin A. F, Control; G, 10 min in brefeldin A;H, 60 min in brefeldin A. Note the rapid redistribution of this marker induced by brefeldin A. Scale bars, 25 μm.
Fig. 6.
Effects of brefeldin A and temperature on the localization of newly synthesized glycoproteins in hippocampal neurons. Fifteen-day-old neurons were pulse-labeled for 1 hr with [3H]mannose at 37°C with or without (control) brefeldin A (5 μg/ml) to block the exit of newly synthesized glycoproteins from the ER. Another group of cells was pulse-labeled with [3H]galactose at 37°C (control) or 20°C to block the exit of proteins from the _trans_-Golgi compartment. The distribution of newly synthesized glycoproteins was evaluated by autoradiography. A, Control cells pulse-labeled with [3H]mannose. B, Cells labeled with [3H]mannose in the presence of brefeldin A. C, Control cells pulse-labeled with [3H]galactose at 37°C. D,E, Cells labeled with [3H]galactose at 20°C. In control cells, newly synthesized glycoproteins were localized in the cell body, dendrites (large arrows), and axons (small arrows). In cells treated with brefeldin A or at 20° C, silver grains concentrate in the cell body and proximal or medial dendrites that were identified by their morphology (B, D) or MAP2 staining (E′). The label was significantly reduced in axons. Scale bar, 50 μm.
Fig. 7.
Immunofluorescent localization of the endomembrane system in hippocampal neurons in culture. Fourteen-day-old neurons were fixed and stained for markers of the ER and GA. A, Localization of the rough ER protein ribophorin I. Note the accumulation of stained ER structures at branch points (asterisks) as well as the absence of label in some dendritic branches (open circles). Axons (open triangles) remained unlabeled. g indicates glial cell. C, Distribution of the medial cisterns of the GA as revealed by α-mannosidase II staining. As is evident, immunostained elements extended into dendrites; indeed, they have a similar subcellular distribution as the immunostaining for the RER. Dendrites (arrows) were identified by double staining for MAP2 (B, D). Scale bars, 50 μm.
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