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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01531

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Tau interaction with microtubules in vivo

¹ Department of Physiology and Biophysics M/C 901, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612, USA
² Department of Psychiatry M/C 901, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612, USA

View larger version (98K): [in a new window]   Fig. 1. GFP-tau binds to individual MTs and induces MT bundling and the formation of neurite-like processes in Xenopus embryo cultures. (A-D) Fluorescence micrographs of fibroblast-like cells expressing GFP-tau. GFP-labeled polymeric structures can be observed in the cytoplasm. Individual polymers at the edge of the cell (marked by arrows in A) are similar in intensity and therefore are likely to represent single MTs. MT bundles can be found both in the soma (B) and inside neurite-like cell processes (C). Panels A and D show representative examples of cells demonstrating non-centrosomal and centrosomal patterns of MT organization. Bar, 10 µm. (E) Series of fluorescence images of the lamella region of a fibroblast transfected with GFP-tau. Both growing (arrows) and shortening (arrowheads) MTs are visible. Time (in seconds) is shown at the upper-right corner. Bar, 10 µm. (F) Quantitative analysis of GFP-tau binding to individual MTs. For each MT, the intensity of fluorescence was measured at distances of 1 µm (open bar) and 5-10 µm (hatched bar) from the tip. The background fluorescence at adjacent MT-free regions was subtracted from the measurements. Data from 50 MTs in seven different neurons are presented as means±s.e.m. (G) Series of fluorescence images of GFP-tau-expressing fibroblasts (elapsed time is stated in seconds). Both individual MTs and MT bundles are detected at the edge of the cell. MT bundles arise through lateral interaction between individual MTs (marked by arrows). Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 2. GFP-tau uniformly labels individual MTs in Xenopus embryo neurons regardless of their location along the axon. (A) Fluorescence image of a neuron expressing GFP-tau. (B,C) Higher-magnification views of the growth cone region (B) and the proximal axon (C) taken from areas marked by rectangles in A. Individual MTs can be resolved in both areas, allowing for direct comparison of tau binding to MTs in different axonal regions. Bar, 10 µm. (D) Quantitative analysis of tau binding to individual MTs at the growth cone and at the proximal axonal segment. Axons chosen for analysis were 150-250 µm in length. The fluorescence intensity of individual MTs was measured at the growth cone region and at the middle axonal segment and, after background subtraction, scaled to adjust for the different levels of tau expression in different neurons. Data are presented as means±s.e.m. Data from 40 MTs in five different neurons.

View larger version (108K): [in a new window]   Fig. 3. Binding of tau to individual MTs depends on MT curvature. (A) Fluorescence micrograph of a fibroblast expressing GFP-tau. Individual MTs can be detected at the lamella region. The boxed regions are shown below at a higher magnification. (B-D) A series of fluorescence images of the lamella of the fibroblast. Images were taken at 3-second intervals. Sites of higher MT curvature (arrows) display higher levels of fluorescence intensity. (E) Fluorescence micrograph of the axonal shaft with a flat growth cone-like protrusion at the lateral edge. The boxed region of the image on the left is shown at higher magnification on the right as a series of images acquired at 3-second intervals. (F) Quantitative analysis of the data for GFP-tau-labeled MTs in fibroblasts (open triangles), GFP-tau-labeled MTs in neurons (stars), and GFP-tubulin-decorated MTs in fibroblasts (filled triangles). Each datum point represents background-subtracted fluorescence intensity of MT at the site of high curvature (I) normalized to that at the straight segment of the same MT (Io), versus the radius of MT curvature (RMT). Data for the cells expressing GFP-tau are grouped together and fitted with a single exponential.

View larger version (85K): [in a new window]   Fig. 4. Tau-MT interaction is highly dynamic. (A-E) Series of fluorescence micrographs of Xenopus fibroblasts (A,C), Xenopus neurons (B,D), and human 356 fibroblasts (E) expressing GFP-tau. The first images were acquired immediately before photobleaching. Individual MTs are marked with arrows in A and B. Elapsed time after photobleaching is shown in seconds at the right corner of each image. The boxed regions mark the approximate location of the bleached zone. The size of the bleached regions is 4 µm. For each time-lapse sequence, the background-subtracted fluorescence intensity of MTs (arbitrary units) was plotted against time in seconds. Fluorescence recovery after photobleaching along individual MTs (A,B,E) is rapid and uniform along MT length. Fluorescence recovery for neurite-like processes produced by fibroblasts (C) and for axons (D) is much slower.

View larger version (34K): [in a new window]   Fig. 5. Recovery of cytoplasmic GFP-tau after photobleaching. (A) Fluorescence images of a GFP-tau-transfected neuron after overnight incubation at 4°C followed by nocodazole treatment (10 µg/ml, 1 hour). The first image was acquired immediately before photobleaching. The time after photobleaching is shown at the upper-right corner of each panel. The size of the bleached zone was 4 µm. No single MTs can be detected with fluorescence microscopy, and therefore fluorescence intensity reflects the concentration of cytoplasmic GFP-tau. Dark elongated structures visible throughout the entire sequence correspond to membrane organelles (most likely mitochondria) which exclude GFP-tau. (B) Fluorescence intensity of the bleached zone (I) normalized to the initial fluorescence (Io) as a function of time.

View larger version (71K): [in a new window]   Fig. 6. Taxol induces rapid dissociation of tau from MTs. (A-F) Fluorescence micrographs of fibroblasts (A,C,D,F) and a neuron (B,E) expressing GFP-tau. Images were taken before exposure to drugs (top images), 3 minutes after application of 1 µM taxol (D,E), and 10 minutes after application of 1 µM vinblastine (F). MTs are clearly visible before drug application (arrows). After incubation with taxol, most of the individual MTs are not decorated with tau; only MT bundles (arrowheads) are seen. Incubation with vinblastine resulted in some MT depolymerization; however, tau is still associated with single MTs (arrows). Bar, 10 µm.

View larger version (145K): [in a new window]   Fig. 7. Brief incubation with taxol does not significantly affect the MT array. Fluorescence micrographs of the same fibroblast-like cell expressing GFP-tau and loaded with Cy3-tubulin before (upper panel) and 3 minutes after (lower panel) the onset of incubation with 1 µM taxol. Exposure to taxol induced rapid retraction of the leading edge and significant change in the overall shape of the cell. Single MTs before exposure to taxol can be seen both in the GFP channel (A) and in the Cy3 channel (B). After incubation with taxol (D), Cy3-labeled MTs are largely intact (arrows). No GFP-tau-labeled MTs can be detected after taxol incubation (C).

View larger version (72K): [in a new window]   Fig. 8. Taxol induces rapid dissociation of tau from MTs in a human cell line. Fluorescence images of a 356 fibroblast expressing GFP-tau. The first image was acquired immediately before application of taxol (1 µM). The time after taxol application is given in seconds. Individual MTs (arrows) and MT bundles (arrowhead) are becoming progressively dimmer, whereas the level of background cytoplasmic fluorescence increases during incubation. Bar, 10 µm.

View larger version (73K): [in a new window]   Fig. 9. Detergent extraction of GFP-tau-expressing cells. Fluorescence images of fibroblasts (A,B) and a neuron (C) expressing GFP-tau before (top images) and after (bottom images) extraction in a buffer containing 10 µM taxol. The time after the onset of extraction is shown in the lower-right corner of each panel. Individual MTs (arrows in A) rapidly lose associated GFP-tau. In contrast, GFP-tau remains associated with MT bundles (arrowheads in B,C) for as long as 30 minutes after extraction. Bars, 10 µm.

View larger version (33K): [in a new window]   Fig. 10. Taxol displaces tau from microtubules in vitro. CHO cells transfected with PcDNA3 (lanes 1 and 1'), wild-type tau (lanes 2 and 2') or GFP-tau (lanes 3 and 3') were homogenized and the supernatants were subjected to conditions allowing the polymerization of tubulin in vitro in the absence (lanes 1,2,3) or presence (lanes 1',2',3') of taxol. Microtubule pellets were examined by SDS-PAGE as described in Materials and Methods. Tau protein and GFP-tau were detected with a polyclonal antibody against the C-terminus of tau (A). The same blots were developed with antibody against -tubulin (B).

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