Article Figures & Data
Figures
- Fig. 1.
Conserved domain structure in mStau protein. An amino acid alignment from rStau (EMBL accession number AJ10200), mouse (EST clones AA106767 and AA896810), human (EST clones AA206573 andR62169) and Drosophila (M69111) is shown. The partial rStau cDNA was cloned by PCR, and its sequence was deposited into EMBL database (EMBL accession number AJ10200). Over the entire 117 amino acid domain, rStau shows 48% identity (71% similarity) compared with the Drosophila sequence. The recent identification of both a human Stau (Marión et al., 1999) and a mouse full- length Stau clone (Wickham et al., 1999) with high homologies toDrosophila Staufen protein verified the sequences predicted from the EST clones. Residues shaded in blackand gray are regions of identity and similarity, respectively. The gray bars indicate the three conserved RBDs, IIa, IIb, and III; the black bar indicates the peptide chosen for immunization within the highly homologous RBD IIa region of Staufen protein.
- Fig. 2.
rStau is a 65 kDa protein. Western blot of rat hippocampal neuronal extracts probed with three different anti-mStau antibodies (lanes 4–6) and its corresponding preimmune antisera (lanes 1–3). Two of the three antisera were monospecific for the 65 kDa band (arrow). The only visible band from one of the preimmune sera was a 72 kDa band in lane 3 (arrowhead). When we compared the expression level of Stau on astrocyte (lane 7) and hippocampal neuronal extracts (lane 8), we found approximately the same signal for mStau. The size of the molecular weight markers is indicated on the left: 202, 104, 82, 66, 48, 33, and 28 kDa. All three antisera gave identical patterns of labeling (data not shown) as the one used in Figures 3 and4.
- Fig. 3.
Intracellular distribution of rStau during the development of polarity in rat hippocampal neurons. mStau labeling is present in the cell body of stage 1 cells (a) and in the cell body and all neurites of stage 2, 3, and 4 (c, e, g, respectively). b, d,f, h, and j show all the processes of the cells identified by α-tubulin. At stage 5 (i), mStau becomes excluded from some of the thin, α-tubulin-positive processes (j,arrowheads). In this experiment, rat hippocampal neurons for 2, 4, and 8 d in culture have been used.
- Fig. 4.
Intracellular distribution of mStau in mature rat hippocampal neurons in culture (14 d in vitro).a, Phase-contrast image of a representative cell.b, mStau labeling is present in the cell body and some of the long processes identified as dendrites with the specific dendritic marker MAP2 (c). Note that axons (arrowheads, MAP2-negative processes) are not labeled with the mStau antibody. d, Phase-contrast image of another representative cell. Double-immunofluorescence of neurons with mStau (e) and the axonal marker tau-1 (f). Note that axons (arrowheads, tau-1-positive processes) are not labeled with the mStau antibody (arrows, a Stau-positive dendrite). The average diameter of a CA1 pyramidal neuron is between 8 and 12 μm.
- Fig. 5.
Postsynaptic localization of Staufen protein and association with MTs and ERs. a, Staufen gold labeling (arrowheads) is exclusively found in dendrites (D) but not axonal terminals (AT) of hippocampal neurons (ratio of 13.4:1; see Results). Some gold clusters were associated with or in close vicinity of tubular ER structures in the dendritic process (38% of all gold; see Results). b, At higher magnification, mStau gold labeling is clearly seen in dendrites but not axonal terminals.c, Staufen labeling was found on bundles of MTs (mt) derived from a dendritic process. The nearby fasciculating axon above the dendrite did not contain any mStau label. Scale bar (in c): a, 250 nm;b, c, 200 nm. m, Mitochondria; sv, synaptic vesicle; t, tubular network; psd, postsynaptic density;mvb, multivesicular body.
- Fig. 6.
Staufen protein is found in large dendritic clusters and colocalizes with rough ER in mature rat hippocampal cells in culture. Immunofluorescence of a saponin-treated cell (a) using anti-mStau antibodies shows mStau in the form of clusters (most likely membrane-bound). b–e, Double-immunofluorescence of neurons with mStau (b,d) and the rough ER marker ribophorin I (c, e) reveals good colocalization between these two proteins (arrowheads andarrows). This confirms data shown in Figure3a and data obtained in HeLa cells (Marión et al., 1999).
- Fig. 7.
Colocalization of mStau with RNPs in hippocampal neurons. RNPs and mitochondria were visualized with the RNA-specific dye SYTO14 and the degree of colocalization with mStau was revealed by confocal microscopy. Immunofluorescence for mStau (a) reveals selective dendritic labeling (arrows). SYTO14 labeling of the same cell (b) additionally stains axons (arrowheads). Because axons do not contain ribosomes or mRNAs, the labeling most likely reflects mitochondria (Knowles et al., 1996). At higher magnification, mStau (c) and SYTO14 label (d) precisely colocalize in many, but not all, clusters in the dendrites. Arrowheadsindicate mStau in SYTO14-labeled RNPs; arrows point to SYTO14 clusters that do not contain mStau. These represent either free mRNA or mitochondria. The average diameter of a CA1 pyramidal neuron is between 8 and 12 μm. Asterisks mark the branching point of a distal dendrite.
Tables
Compartment # of gold particles (mean ± SEM) % of gold particles (mean ± SEM) Dendrites 13.4 ± 1.5 94 ± 2% Axons 1.0 ± 0.4 6 ± 2% Staufen association with Intracellular membranes 52 of 134 38 ± 4.5% Microtubules 30 of 32 96 ± 4.2% Random electron micrographs were chosen for this quantitation. In the first row, gold particles within 10 axons and dendrites were counted and expressed as average numbers of gold particles. In the second row, numbers of gold particles were listed that were found to be associated with intracellular membranes (most likely ERs) and MTs.
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