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First published online 21 September 2005
doi: 10.1242/jcs.02558

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Molecular motors implicated in the axonal transport of tau and -synuclein

Department of Neuroscience, King's College London, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK

View larger version (37K): [in a new window]   Fig. 1. Tau and -synuclein transport depends on an intact microtubule cytoskeleton. (A) Rat cortical neurons were either not treated or treated with cytochalasin B to disrupt the actin cytoskeleton or with nocodazole to depolymerise microtubules. Tubulin was visualised with antibody to -tubulin and FITC-conjugated secondary antibody (green) and actin was detected with phalloidin-TRITC (red). Merged images of tubulin and actin are shown on the right. Bar, 10 µm. (B,C) Neurons were transfected with -synuclein (B) or tau-EGFP (C) and cells were fixed at 30-minute intervals after transfection. Neurons were either not treated () or treated with nocodazole 3 hours after transfection () or cytochalasin B (). Neurons transfected with -synuclein were immunostained for -synuclein using 90 antibody and tau was detected by direct fluorescence of EGFP. The distance travelled by the exogenous protein was measured from the perimeter of the cell body along the axon to the limit of the fluorescent front. Each point represents the mean±s.e.m. (n=60-130 measurements). Treatment with nocodazole inhibited the transport of both proteins (*P<0.01 using one-way ANOVA), whereas transport of neither of the proteins was affected by treatment with cytochalasin B (P>0.05), indicating that an intact microtubule network is essential for the transport of tau and -synuclein.

View larger version (65K): [in a new window]   Fig. 2. Tau- and -synuclein-containing structures move at rates comparable with fast transport. (A,B) Rat cortical neuron transfected with 0N4Rtau-EGFP, 48 hours after transfection. The movement of tau-containing structures were monitored using live image analysis. The frames, taken at the indicated time intervals in seconds, show live images of tau-containing structures moving at a rate comparable with that of fast transport. The frames show overall anterograde (A) and retrograde (B) movement of a tau-containing structure (*). See also supplementary material Movies 1 and 2. (A',B') Tracking of the tau-containing structures shown in A and B. (C,D) As A and B, showing overall anterograde (C) and retrograde (D) movement of -synuclein-containing particles. See also supplementary material Movies 3 and 4. (C',D') Tracking of the -synuclein-containing particles shown in C and D. Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. Tau-EGFP and -synuclein-EGFP-containing structures colocalise with kinesin-1. E18 rat cortical neurons were transfected with tau-EGFP or -synuclein-EGFP, fixed after 48 hours, immunostained for kinesin-1 and visualised by laser confocal microscopy. The merged images show yellow areas (arrows) that indicate colocalisation of tau or -synuclein with kinesin-1 immunoreactivity. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 4. Tau and -synuclein each co-immunoprecipitate with kinesin-1, whereas only -synuclein co-immunoprecipitates with dynein. Rat brain homogenate was immunoprecipitated using antibodies recognising tau (left panels) or -synuclein (right panels). Immunoprecipitates were analysed on western blots with antibodies to kinesin-1 (upper panels) or dynein (lower panels). The tau antibody co-immunoprecipitated kinesin-1 but not dynein. The antibody to -synuclein co-immunoprecipitated both kinesin-1 and dynein. Molecular mass markers are shown on the left (kDa).

View larger version (43K): [in a new window]   Fig. 5. GST-tau and GST--synuclein each interact with protein complexes containing kinesin-1, whereas only GST--synuclein interacts with protein complexes containing dynein. Western blots of rat brain homogenate incubated with GST, GST-tau or GST--synuclein bound to glutathione-Sepharose beads. Bound proteins were analysed on western blots with antibodies against kinesin-1 (A, B) or dynein (C,D). Panels A and B show an interaction of GST-tau and GST--synuclein with kinesin-1 in brain homogenate, respectively. There is no detectable interaction between GST-tau and dynein (C), whereas GST--synuclein interacts with dynein (D). No interactions were detected in brain homogenate incubated with purified GST alone.

View larger version (37K): [in a new window]   Fig. 6. Tau interacts with GST-KLC1 and GST-KLC2. (A,B) Western blots of rat brain homogenate (A) or recombinant human tau (B) probed with a polyclonal antibody to tau after incubation with purified GST, GST-KLC1 or GST-KLC2 bound to glutathione-Sepharose beads. Both recombinant human tau and endogenous rat brain tau, or complexes containing rat brain tau, bind to GST-KLC1 and GST-KLC2, but not to purified GST.