Fig. 2.

Microtubule nucleation is disrupted in the presence of aggresomesin undifferentiated and differentiated SH-SY5Y cells. (Ai–iv) SH-SY5Y cells have an extensive microtubule network. Upon cold treatment (4°C, t=0), microtubules depolymerise. Upon warming, microtubules nucleate from the centrosome forming a characteristic aster, which continues to grow until the network is re-established. In SH-SY5Y cells the aster is seen within in 30 s (37°C, t+0.5) and the microtubule network is re-established within 10 min (37°C, t+10). (Av–viii) In the presence of aggresomes formed by the overexpression of α-syn (GFP-fusion), the centrosome is unable to re-establish the network in 10 min. (Aix–xvi) The same result is achieved by overexpression of α-syn A30P and A53T familial mutants. (Axvii–xx) In the presence of aggresomes formed after MG132 treatment, the centrosome is unable to nucleate microtubules to re-establish this network. (Axxi–xxiv) In differentiated SH-SY5Y (tyrosine hydroxylase, TH, in green), microtubule nucleation is seen as asters from the centrosome (arrow heads and inset). (Axxv–xxviii) In the presence of aggresomes the density of the network is reduced and microtubule nucleation is severely compromised. (B) Quantification of microtubule regrowth in experiments in A [P=0.0001, one-way ANOVA (for α-syn over-expression), 100 cells, n=3 replicates; P=0.0001, by Student's t-test (MG132 experiments), 100 cells, n=3 replicates]. Microtubule nucleation and re-establishment of this network was quantified by scoring cells (yes or no) whether the network was re-established in 10 min. Microtubule staining for α-syn experiments shown separately in Fig. S1 together with GFP control. Scale bars: 10 μM.