Fig. 2

The C terminus of alpha-synuclein binds to synaptic vesicles upon calcium binding. a CEST-NMR experiments were performed on alpha-synuclein and synaptic vesicles in the absence (black) or presence of calcium (red, 6 mM). In the absence of calcium, the N terminus shows the strongest interaction with synaptic vesicles. Upon addition of calcium, the interaction of the C terminus and also of some residues of the NAC-region increases, which is seen as a reduction of the signal. Experiments were repeated twice. b Lipid pull-down experiment showing the transient nature of alpha-synuclein lipid binding. Western blot of the amount of alpha-synuclein pulled down by the lipids showing that calcium-induced lipid binding of alpha-synuclein is reversible upon addition of the calcium chelator EGTA. *p = 0.0263, calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 6 for control and CaCl₂, n = 4 for EGTA and CaCl₂ + EGTA, data from three biological repeats, d.f. 16. c dSTORM super-resolution imaging of alpha-synuclein and VAMP2 on isolated synaptosomes displaying alpha-synuclein clustering under normal physiological conditions with 2.5 mM calcium in the extracellular buffer (upper panel). Upon calcium depletion in the extracellular buffer, using 1 mM EGTA, alpha-synuclein localization was dispersed (lower panel). d Cluster analysis of alpha-synuclein and VAMP2 immunostaining showing increased cluster size of alpha-synuclein upon calcium depletion, whereas VAMP2 cluster size is the same either in the presence of calcium or upon calcium depletion in the extracellular buffer. ****p < 0.0001, ^nsp = 0.6363 calculated using two-tailed t-test, graphs indicate mean ± s.e.m. N = 22 for +Ca²⁺ and n = 30 for −Ca²⁺, where n indicates single synaptosomes, data form three biological repeats, d.f. 50