Recent developments in image processing have greatly advanced our understanding of biomolecular processes in vitro and in vivo. In particular, using Gaussian models to fit the intensity profiles of nanometer-sized objects have enabled their two-dimensional localization with a precision in the one-nanometer range. Here, we present an algorithm to precisely localize curved filaments whose structures are characterized by subresolution diameters and micrometer lengths. Using surface-immobilized microtubules, fluorescently labeled with rhodamine, we demonstrate positional precisions of ∼2 nm when determining the filament centerline and ∼9 nm when localizing the filament tips. Combined with state-of-the-art single particle tracking we apply the algorithm 1), to motor-proteins stepping on immobilized microtubules, 2), to depolymerizing microtubules, and 3), to microtubules gliding over motor-coated surfaces.

Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation or, in the presence of opposing motors, generate oscillatory movements. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule-microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular off-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization.

The stepping behavior of single kinesin-1 motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in small groups when they transport cellular cargo. Until now, the cooperative interactions between motors in such groups were poorly understood. A fundamental question is whether two or more motors that move the same cargo step in synchrony, producing the same step size as a single motor, or whether the step size of the cargo movement varies. To answer this question, we performed in vitro gliding motility assays, where microtubules coated with quantum dots were driven over a glass surface by a known number of kinesin-1 motors. The motion of individual microtubules was then tracked with nanometer precision. In the case of transport by two kinesin-1 motors, we found successive 4-nm steps, corresponding to half the step size of a single motor. Dwell-time analysis did not reveal any coordination, in the sense of alternate stepping, between the motors. When three motors interacted in collective transport, we identified distinct forward and backward jumps on the order of 10 nm. The existence of the fractional steps as well as the distinct jumps illustrate a lack of synchronization and has implications for the analysis of motor-driven organelle movement investigated in vivo.collective motion ͉ microtubules ͉ nanometer tracking ͉ quantum dots A ctive cellular transport, such as organelle traffic, is driven by motor proteins of different families such as kinesin, myosin, and cytoplasmic dynein (1). On the level of single molecules, the stepping behavior of these motors has been studied in great detail in vitro (2-4). Although many motors within these families have been shown to be processive, meaning that individual motors are able to produce continuous motion (5-7), in cells, they usually operate in small groups (8-11). However, little is known about the possible mechanisms of coordination between such motors.Kinesin-1 (henceforth denoted ''kinesin'') is a heterotetrameric motor enzyme that converts the chemical energy of ATP hydrolysis into mechanical work. A single motor moves with 8-nm steps (12) toward the plus end of microtubules (MTs). It translocates with an asymmetric hand-over-hand mechanism (13, 14) and performs Ϸ100 steps before detaching from the MT (5, 15, 16). The step size is independent of the force and the ATP concentration (17). When a small group of kinesins carries the same cargo, it is known that the run length increases with the number of motors (5,15,18). But what is the step size of such cargo movement? A number of recent studies reported the detection of 8-nm steps in the displacement of cellular cargo in vivo (19,20). Although no detailed mechanism for the occurrence of these 8-nm steps, which correspond to the unit step size of single motor molecules, was elucidated, these findings are intriguing as they might suggest a synchronization of the motors (i.e., all of them stepping at the same time) involved in transport.When studyin...