Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the kinesin-8 Kip3 and the kinesin-13 MCAK. Surprisingly, we found that, even in the absence of the kinesins, the microtubule catastrophe frequency depends on the age of the microtubule, indicating that catastrophe is a multistep process. Kip3 slowed microtubule growth in a length-dependent manner and increased the rate of aging. In contrast, MCAK eliminated the aging process. Thus, both kinesins are catastrophe factors; Kip3 mediates fine control of microtubule length by narrowing the distribution of maximum lengths prior to catastrophe, whereas MCAK promotes rapid restructuring of the microtubule cytoskeleton by making catastrophe a first-order random process.

Motor proteins in the kinesin-8 family depolymerize microtubules in a length-dependent manner that may be crucial for controlling the length of organelles such as the mitotic spindle. We used single-molecule microscopy to understand the mechanism of length-dependent depolymerization by the budding yeast kinesin-8, Kip3p. We found that after binding at a random position on a microtubule and walking to the plus end, an individual Kip3p molecule pauses there until an incoming Kip3p molecule bumps it off. Kip3p dissociation is accompanied by removal of just one or two tubulin dimers (on average). Such a cooperative mechanism leads to a depolymerization rate that is proportional to the flux of motors to the microtubule end and accounts for the length dependence of depolymerization. This type of feedback between length and disassembly may serve as a model for understanding how an ensemble of molecules can measure and control polymer length.

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Animals continuously gather sensory cues to move towards favourable environments. Efficient goal-directed navigation requires sensory perception and motor commands to be intertwined in a feedback loop, yet the neural substrate underlying this sensorimotor task in the vertebrate brain remains elusive. Here, we combine virtual-reality behavioural assays, volumetric calcium imaging, optogenetic stimulation and circuit modelling to reveal the neural mechanisms through which a zebrafish performs phototaxis, i.e. actively orients towards a light source. Key to this process is a self-oscillating hindbrain population (HBO) that acts as a pacemaker for ocular saccades and controls the orientation of successive swim-bouts. It further integrates visual stimuli in a state-dependent manner, i.e. its response to visual inputs varies with the motor context, a mechanism that manifests itself in the phase-locked entrainment of the HBO by periodic stimuli. A rate model is developed that reproduces our observations and demonstrates how this sensorimotor processing eventually biases the animal trajectory towards bright regions.

Friction limits the operation of macroscopic engines and is critical to the performance of micromechanical devices. We report measurements of friction in a biological nanomachine. Using optical tweezers, we characterized the frictional drag force of individual kinesin-8 motor proteins interacting with their microtubule tracks. At low speeds and with no energy source, the frictional drag was related to the diffusion coefficient by the Einstein relation. At higher speeds, the frictional drag force increased nonlinearly, consistent with the motor jumping 8 nanometers between adjacent tubulin dimers along the microtubule, and was asymmetric, reflecting the structural polarity of the microtubule. We argue that these frictional forces arise from breaking bonds between the motor domains and the microtubule, and they limit the speed and efficiency of kinesin.

SummaryThe vestibular apparatus provides animals with postural and movement-related information that is essential to adequately execute numerous sensorimotor tasks. In order to activate this sensory system in a physiological manner, one needs to macroscopically rotate or translate the animal’s head, which in turn renders simultaneous neural recordings highly challenging. Here we report on a novel miniaturized, light-sheet microscope that can be dynamically co-rotated with a head-restrained zebrafish larva, enabling controlled vestibular stimulation. The mechanical rigidity of the microscope allows one to perform whole-brain functional imaging with state-of-the-art resolution and signal-to-noise ratio while imposing up to 25° in angular position and 6,000°/s2 in rotational acceleration. We illustrate the potential of this novel setup by producing the first whole-brain response maps to sinusoidal and stepwise vestibular stimulation. The responsive population spans multiple brain areas and displays bilateral symmetry, and its organization is highly stereotypic across individuals. Using Fourier and regression analysis, we identified three major functional clusters that exhibit well-defined phasic and tonic response patterns to vestibular stimulation. Our rotatable light-sheet microscope provides a unique tool for systematically studying vestibular processing in the vertebrate brain and extends the potential of virtual-reality systems to explore complex multisensory and motor integration during simulated 3D navigation.

Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.

During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.