Abstract
Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.
Highlights
Motor proteins from the kinesin- and dynein-superfamilies fulfill essential mechano-chemical functions in eukaryotic cells
Guanylyl-(α,β)-methylene-diphosphonate (GMP-CPP) speckled MTs (S-MTs) were grown in a two-step process. 25μl of BRB80 solution (80mM Pipes [solution consisting of 0.5mg/ml casein (Sigma)], pH 6.9, with KOH [VWR], 1mM EGTA [Sigma], 1mM MgCl2 [VWR]) supplemented by a high concentration (12μM) of porcine tubulin (S-MTs: 99.44% unlabeled and 0.56% rhodamine-labeled; biotinylated S-MTs [B-S-MTs]: 94.44% unlabeled, 5% unlabeled biotinylated (Cytoskeleton Inc., Denver, CO) and 0.56% rhodamine-labeled), 1 mM GMP-CPP (Jena Bioscience, Jena, Germany) and 4mM MgCl2 were incubated on ice for 5min and for 20-30min at 37°C to grow a large number of short, dimly-labeled MT seeds
The resulting intensity profiles of the speckles showed a periodic variation in the intensity (Fig 1E and S1 Fig). (v) Performing an auto-correlation on the intensity profile for individual speckles (Fig 1F and S1 Fig) and subsequently obtaining the power spectral density (PSD) of the autocorrelation data yields the peaks corresponding to the spatial frequencies in the intensity signal for the speckles
Summary
Motor proteins from the kinesin- and dynein-superfamilies fulfill essential mechano-chemical functions in eukaryotic cells. The one-dimensional motion of these motors along microtubules (MTs) has been studied in great detail using in vitro stepping motility assays, for example by tracking single fluorescently labeled motors [1,2] as well as motors coupled to microbeads [3,4,5], quantum dots (QDots) [6,7,8] or DNA origami [9,10]. MTs are three-dimensional, cylindrical structures (diameter of 25nm), which consist of about 13 adjacent protofilaments forming a parallel array of tracks. While some kinds of processive motors follow the axes of individual protofilaments, others take stochastic off-axis steps potentially with or without bias in one specific direction. One simple experimental method to distinguish between these modes of movement is based on in vitro gliding motility assays where MTs glide
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