Volker Bormuth

Kinesin-8 Motors Act Cooperatively to Mediate Length-Dependent Microtubule Depolymerization

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.

Protein Friction Limits Diffusive and Directed Movements of Kinesin Motors on Microtubules

Friction in Microscopic Motor Friction arises because adhesive bonds between two bodies must be broken in order for them to move relative to each other. Now Bormuth et al. (p. 870; see the Perspective by Veigel and Schmidt) have used single molecule measurements to characterize the frictional drag force of kinesin-8 motor proteins interacting with their microtubule track. Friction, arising from rupture of bonds with the track, constrains the speed and efficiency of the motor protein. Rupture of bonds between a molecular machine and its track creates friction, which constrains speed and efficiency. 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.

Depolymerizing Kinesins Kip3 and MCAK Shape Cellular Microtubule Architecture by Differential Control of Catastrophe

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.

Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy

In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

Injection and flow control system for microchannels

In spite of considerable efforts, flow control in micro-channels remains a challenge owing to the very small ratio of channel/supply-system volumes, as well as the induction of spurious flows by extremely small pressure or geometry changes. We present here an inexpensive and robust system for flow control in a microchannel system, based on a dynamic control of reservoir pressures at the end of each channel. This system allows flow equilibration with a time constant smaller than one second, and is also able to maintain stable flux from stopped flow to many microl min(-1) range over several hours. It is robust to changes in ambient pressure and temperature. This system further includes a feature for sub-microliter sample injection during the experiment. We quantify flow control in elastomer and thermoplastic channels, and demonstrate the impact on one application of the system, namely the reproducible, automated separation of large DNA by electrophoresis in a self-organized magnetic bead matrix in a microchannel.

LED illumination for video-enhanced DIC imaging of single microtubules

In many applications high‐resolution video‐enhanced differential interference contrast microscopy is used to visualize and track the ends of single microtubules. We show that single ultrabright light emitting diodes from Luxeon can be used to replace conventional light sources for these kinds of applications without loss of function. We measured the signal‐to‐noise ratio of microtubules imaged with three different light emitting diode colours (blue, red, green). The blue light emitting diode performed best, and the signal‐to‐noise ratios were high enough to automatically track the ends of dynamic microtubules. Light emitting diodes as light sources for video‐enhanced differential interference contrast microscopy are high performing, low‐cost and easy to align alternatives to existing illumination solutions.

Kinesin-8 is a low-force motor protein with a weakly bound slip state.

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.

The Highly Processive Kinesin-8, Kip3, Switches Microtubule Protofilaments with a Bias toward the Left

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.

Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays

Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as nonprocessive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.