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doi: 10.1242/10.1242/jcs.00058

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Visualization of translated tau protein in the axons of neuronal P19 cells and characterization of tau RNP granules

Department of Neurobiology, The Weizmann Institute of Science, Rehovot, 76100 Israel

View larger version (60K): [in a new window]   Fig. 3. HuD, KIF3A and tau mRNA colocalize in RNP granules. (a) Phase image: the growth cone is shown on the right side of the panel. Colocalization of tau mRNA (b, red), HuD protein (c, green) and KIF3A (d, cyan) to yield the merged image presented in e of the axon and growth cone of differentiated P19 cells. The curved arrowhead denotes colocalization of the three components (white). The asterisk denotes colocalization of HuD and KIF (light green). The straight arrowhead denotes colocalization of HuD and tau mRNA (yellow). Bar, 1 µm (x40,000 magnification).

View larger version (59K): [in a new window]   Fig. 1. HuD protein colocalizes with tau mRNA in RNP granules. (A) Confocal image analysis of HuD protein localization in differentiated P19 cells. (a) Phase image of a differentiated P19 cell. (b) Expression of GFP-tau protein. (c) Staining with HuD antibodies. (d) In situ hybridization analysis with the GFP probe (which detects the transfected tau mRNA). (e) Merged confocal image of b, c and d (x2500 magnification). The solid arrowheads denote axons; empty arrowheads denote dendrites; and small solid curved arrowheads denote neuronal cell bodies. Bar, 10 µm. (B) Confocal merged image of a growth cone. An enlarged merged image from the growth cone of the cell shown in A. The merged staining includes the GFP-tau fluorescence (green), HuD immunostaining (cyan) and GFP in situ hybridization (red) to yield a white area of colocalization. Aggregation of tau granules is marked by a solid white arrowhead; free HuD protein (cyan) is marked by an asterisk. Bar, 1 µm (x20000 magnification).

View larger version (41K): [in a new window]   Fig. 2. KIF3A protein colocalizes with tau mRNA in RNP granules. (A) Confocal image analysis of KIF3A protein localization in differentiated P19 cells. (a) Phase image of differentiated P19 cell. (b) Expression of GFP-tau protein. (c) Staining with KIF3A antibodies. (d) In situ hybridization analysis with the GFP probe (which detects the transfected tau mRNA). (e) Merged confocal image of b, c and d (x2500 magnification). The solid arrowheads denote axons, empty arrowheads denote dendrites and small solid curved arrowheads denote neuronal cell bodies. Bar, 10 µm. (B) Confocal merged image of a growth cone. The enlarged merged image from the cell shown in A. The merged staining includes the GFP-tau fluorescence (green), KIF3A immunostaining (cyan) and GFP in situ hybridization (red), yielding a white colocalization. Aggregation of tau granules is marked by a solid arrowhead. A non-localized KIF3A signal (cyan) is marked by an asterisk. Bar, 1 µm (x20000 magnification).

View larger version (53K): [in a new window]   Fig. 4. (A) A field view of differentiated P19 cells treated with KIF3A antisense ODN, analyzed using a confocal micoscope. (a,b) Control untreated cells; (c,d) treated cells; (a,c) GFP-tau fluorescence. (b,d) KIF3A protein localization visualized by immunohistochemical staining with KIF3A antibodies. Bar, 20 µm. (B) Immunoblot analysis of cell extracts prepared from control-differentiated P19 cells or from cells treated with KIF3A antisense ODN and KIF3A sense ODN. The blots were reacted with KIF3A, KIF1A, tau, tubulin, MAP2, synaptophysin and neurofilament antibodies. (This is a representative blot from three experiments.)

View larger version (45K): [in a new window]   Fig. 5. Confocal image analysis of a single differentiated P19 cell treated with KIF3A antisense ODN. (a-e) Differentiated untreated P19 cells. (f-j) KIF3A antisese ODN-treated cell. (k-o) KIF3A sense ODN-treated cell. (a,f,k) Localization of KIF3A protein, as visualized by staining with anti-KIF3A antibodies. (b,g,l) Expression of GFP-tau protein. (c,h,m) In situ hybridization with the GFP probe (which detects the transfected tau mRNA). (d,i,n) Localization of tubulin protein, as visualized by staining with tubulin antibodies. (e,j,o) In situ hybridization with tubulin. Solid arrowheads denote axons, empty arrowheads denote dendrites and small solid curved arrowheads denote neuronal cell bodies. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 6. Co-immunoprecipitation of HuD, KIF3A and tubulin from extracts prepared from P19 differentiated cells. (A) Immunoprecipitated complexes analyzed by immunoblotting with HuD antibody. (B) Immunoprecipitated complexes analyzed by immunoblotting with KIF3A antibody. Non-immune serum (NIS) was used as a control for specific immunoprecipitation.

View larger version (84K): [in a new window]   Fig. 7. Confocal image analysis of differentiated living P19 cell lines treated with emetine, a protein-synthesis inhibitor. (Aa,b) A control differentiated cell. The insert shows the enlargement of the growth cone region. (c,d) GFP-tau protein fluorescence in an emetine-treated cell. (e,f) GFP-tau protein fluorescence in a cell following a recovery period of 3 hours in fresh media. (B) Tau mRNA is found in emitine treated cells. (a) Phase view of a treated cell, which shows no GFP fluorescence (b). (c) In situ hybridization with GFP probe. Solid arrowheads denote axons, empty arrowheads denote dendrites and small solid curved arrowheads denote neuronal cell bodies. Bar, 10 µm.

View larger version (13K): [in a new window]   Fig. 8. Local GFP tau synthesis in the axon of P19 differentiated cell during the recovery from emetine treatment. (a) Phase image. (b,e) Recovery periods of 1.5, 2, 2.5 and 3 hours. The cell bodies are circled. Bar, 10 µm.