Axonal tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal

Abstract

Subcellular mRNA localization, a fundamental mechanism for regulating gene expression, leads to local protein translation that results in the generation of neuronal cell polarity. In this study, we have used P19 embryonic carcinoma cells, which are amenable to transfection, and selection of clonal stable cell lines that are not overexpressing the constructs. We identified the 3' untranslated region (3'UTR) tau axonal localization signal and examined its effect on tau protein localization in nondifferentiated and neuronally differentiated P19 cells. Using GFP-tagged tau constructs combined with in situ hybridization analysis, we demonstrated colocalization of the targeted tau mRNA and its translated protein in the axon and growth cone. Absence of or mutation in the 3'UTR axonal targeting region of tau mRNA resulted in suppression of tau mRNA localization, and both tau mRNA and tau protein remained in the cell body. Swapping between the 3'UTR tau mRNA axonal localization signal and the 3'UTR MAP2 mRNA dendritic targeting signal proved that the localization of the proteins into the axon or dendrites depends on the specific 3'UTR targeting signals. Moreover, the identification of ribosomal proteins in the axon lends further support to the presence of protein synthetic machinery in the axons, a prerequisite for local translation. It is suggested therefore that the P19 cell system can be used to analyze mutations that affect mRNA transport and local translation and that it has the potential of being used to examine the onset of the neuronal differentiation process.

Fig. 1.

Confocal image analysis of undifferentiated P19 living cell lines. a, Field view of a selected stable cell line transfected with GFP-tau–cod-H construct. b, Phase view of a. Because the photograph shows living cells, not all of the cells in the field are in the same plane; hence, the differences observed in the intensity of GFP fluorescence. Scale bar, 50 μm. c, Distribution of tau protein on MTs in the stable cell line transfected with the GFP-tau–cod-H construct. d, Distribution of P19 cells transiently transfected with the GFP vector alone. Scale bar, 5 μm.

Fig. 2.

Confocal image analysis of MAP segregation in neuronally differentiated P19 cells. a–d, P19 stable cell expressing GFP-tau–cod-H construct, neuronally differentiated for 9 d. MAP segregation was analyzed by confocal microscopy. Scale bar, 5 μm. a, GFP-tau protein is visible (green) in the soma and axon of a single cell.b, MAP-2 is visible (red) in the soma and dendrite of the same cell. c, Computer-merged analysis of GFP-tau (green) and MAP-2 (red) proteins; GFP-tau and MAP-2 colocalize (orange) in the soma but segregate to the axon and the dendrite, respectively.d, Phase view of the same P19 cell. e, The same cell stained with tau-1 antibodies. Large filled arrowheads denote an axon, large open arrowheads denote dendrites, and small filled arrowheads denote a neuronal cell body.

Fig. 3.

Confocal image analysis of neuronally differentiated P19 living cell lines stably transfected with GFP-tau constructs. Distribution of GFP-tau in cell lines transfected with GFP-tau–cod-H construct (a, b), GFP-tau–cod construct (c, d), and GFP-tau–cod-Hdel (e,f) in differentiated P19 cells. Theright panels show a phase view of the cells. Scale bars, 20 μm. Large filled arrowheads denote an axon,large open arrowheads denote dendrites, and small filled arrowheads denote a neuronal cell body.

Fig. 4.

Confocal microscopy image of P19 cell cultures analyzed by in situ hybridization combined with immunohistochemistry using tubulin antibodies. a–d, Confocal image of a P19 cell line transfected with GFP-tau–cod-H construct. e–h, Confocal image of a P19 cell line transfected with GFP-tau–cod construct. i–l, Confocal image of a P19 cell line transfected with GFP-tau–cod-Hdel construct. a, e, i, GFP-tau fluorescence. b, f,j, P19 cell immunostained with tubulin antibodies.c, g, k, In situ hybridization using a GFP probe labeled with UTP-dig and detected with anti-dig HRP/Cy5. d, h,l, Merged confocal image showing colocalization of GFP-tau fluorescence with tubulin and with GFP-tau mRNA. Scale bars, 10 μm. Large filled arrowheads denote an axon,large open arrowheads denote dendrites, and small filled arrowheads denote a neuronal cell body.

Fig. 5.

Granules observed in the axon and growth cone of a differentiated P19 cell. Merged confocal image at higher magnification of the axon and growth cone analyzed by in situhybridization and tubulin staining of a P19 cell line transfected with GFP-tau–cod-H construct (as shown in Fig. 4). Scale bar, 50 μm.Large arrowhead denotes granules in the growth cone, andsmall arrowheads denote granules along the microtubules.

Fig. 6.

Ribosomal proteins are detected in the axon and dendrites of differentiating P19 cells. A, Confocal microscopy image analysis of P19 cell stained with anti-tubulin (a). b, Anti 60s ribosomal proteins. c, In situ hybridization using a tau probe, which detects both the endogenous and transfected tau mRNA. d, Merged confocal image showing colocalization.B, The region boxed in the inset ind is shown in enlarged scale in a′,b′, c′, and d′, corresponding to a, b, c, and d, respectively. Scale bars, 10 μm.Large filled arrowheads denote an axon, large open arrowheads denote dendrites, and small filled arrowheads denote a neuronal cell body.

Fig. 7.

Western blot analysis of GFP-tau proteins in stable P19 cell lines. Expression of GFP-tau proteins detected using tau-1 antibody in undifferentiated P19 cells (a) and in P19 cells after differentiation for 8 d (b). The same membrane was used for detection of tubulin as an internal control (a representative blot).c, Quantitative analysis of the results of four experiments, expressed in arbitrary units calibrated to the signal of cells transfected with GFP-tau–cod construct (100%). Values are means ± SEM. Asterisks mark a significant difference in tau protein levels (*p < 0.05, **p < 0.01).

Fig. 8.

RT-PCR analysis of GFP-tau mRNA levels in stable transfected P19 cell lines. Expression of GFP-tau mRNA levels in undifferentiated P19 cells (a) and in P19 cells after differentiation for 8 d (b). A GAPDH signal was used as an internal control. PCR was allowed to proceed for 25, 30, and 35 cycles using GFP-tau primers (yielding a fragment of 510 bp) and for 20, 25, and 30 cycles using GAPDH primers (yielding a fragment of 340 bp). A representative blot from four experiments is shown. The results demonstrate a linear relationship along the cycles. A control experiment, using RNA isolated from nontransfected P19 cells and processed concomitantly, is presented.

Fig. 9.

MAP2-targeting signal drives tau expression into the dendrites of differentiated P19 cells. Confocal image of a P19 cell line transfected GFP-tau–cod-MAP2-targeting signal. a, Localization of GFP-tau protein in the dendrites. b, P19 cell stained with MAP2 antibodies to visualize the dendrites.c, Localization by in situ hybridization of GFP-tau mRNA with a GFP probe detected with anti-dig HRP/Cy5.d, Merged image of a, b, and c showing the colocalization of GFP-tau protein and mRNA in the dendrites. e, Phase view. Note that the axon seen in e is not stained in a–d because tau is driven to the dendrites. Scale bar, 10 μm. Large filled arrowheads denote an axon, large open arrowheads denote dendrites, and small filled arrowheads denote a neuronal cell body.

Fig. 10.

Tau-targeting signal drives MAP2 expression into the axon of differentiated P19 cells. Confocal image of a P19 cell line transfected with a construct containing GFP-MAP2-cod-fragment-H of tau 3′UTR. a, Localization of GFP-MAP2 protein in the axons.b, P19 cell stained with MAP2 antibodies shows the dendrites and axon. c, Localization by in situ hybridization of GFP-MAP2 mRNA with a GFP probe detected with anti-dig HRP/Cy5. d, Merged image ofa, b, and c showing the colocalization of GFP-MAP2 protein and mRNA in the axon.e, Phase view. Scale bar, 10 μm. Large filled arrowheads denote an axon, large open arrowheadsdenote dendrites, and small solid arrowheads denote a neuronal cell body.