Dyelight 488

Differentiated human dopaminergic neurons were fixed by using 4% paraformaldehyde for 15 min and permeabilised by Triton X-100 at 0.04% as described before^{28 (link)}. Primary antibodies against human aSYN (Santa Cruz C20; Santa Cruz Biotechnology, Dallas, USA), β3-tubulin (Invitrogen, Karlsruhe, Germany) at a concentration of 1:200 were incubated overnight at 4 °C, followed by secondary anti-rabbit, anti-mouse antibodies coupled to fluorophore Dyelight 488, and Dyelight 649 (Invitrogen) at a concentration of 1:500. Images were obtained by using a CCD camera (DFC 360, FX, Leica, Wetzlar, Germany) connected to an epifluorescent microscope (DMI 6000 B, Leica). For co-localisation studies, cells were co-stained with MitoTracker Deep Red (200 ng/ml) for mitochondria and DAPI (4′,6-diamidino-2-phenylindole, 1 µg/ml) for the nucleus. Quantification and analysis of neuronal network was performed by using the Image J software.

Myosin II was purified from
rabbit skeletal muscle and fluorescently labeled with DyeLight 488
(Invitrogen, Carlsbad, CA, USA) according to Alvarado and Koenderink.^{66 (link)} Labeled
and unlabeled myosin II were stored separately in myosin storage buffer
(300 mM KCl, 25 mM KH₂PO₄, 0.5 mM DTT, 50% (v/v)
glycerol, pH 6.5), where the high ionic strength prevents myosin self-assembly
into bipolar filaments. For experiments, myosin II was dialyzed overnight
in glycerol-free myosin buffer (300 mM KCl, 20 mM imidazol, 4 mM MgCl₂, 1 mM DTT, pH 7.4) and controlled self-assembly into bipolar
filaments was induced by adjusting a KCl concentration of 50 mM via
mixing with myosin polymerization buffer (20 mM imidazol, 1.6 mM MgCl₂, 1 mM DTT, 1.2 mM Trolox, pH 7.4). After an incubation time
of 10 min at 20 °C, the bipolar myosin II filaments were immediately
used for contractile experiments. For the contraction experiments,
the F-actin networks were transferred into an actomyosin buffer by
a 10-fold buffer exchange (50 mM KCl, 20 mM imidazol, 2 mM MgCl₂, 1 mM DTT, 1 mM Trolox, pH 7.4). The reorganization of the
networks was performed at a final ATP concentration of 0.1 mM combined
with an ATP-regeneration system of creatine phosphate (10 mM)/creatine
kinase (0.1 mg/mL)^{66 (link)} and a myosin II concentration
of 0.4 μM.