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Jannasch, A.

Publications and source records attributed to Jannasch, A..

5 recordsLinked to original sources

Coin-tossing by Kinesin-1 Head and Tail Binding Collectively Drives Microtubule Patterns

Intracellular microtubule-based transport depends on an essential plus-ended molecular motor, kinesin-1. The N-terminal ATP-dependent head driving motility and a C-terminal cargo interacting tail, both bind microtubules. Previously, the interplay of both domains were hypothesized to play a role in collective microtubule sliding patterns, but the mechanisms remained unclear. Here, we show that full-length Drosophila kinesin-1 generates striking spatiotemporal patterns in gliding assays including bending, looping, and oscillations as well as stop-and-go motion of filaments. In presence of the motor domain alone these patterns are absent, but with the tail only construct, microtubules bind passively, with the tail acting as a static anchor. An equimolar mixture of these two domain constructs reproduces these spatio-temporal patterns both in terms of increased bending with length and velocity distributions. Our results support a simple coin-toss model, where either head or tail bind microtubules with equal probability, leading to antagonistic interactions that drives emergent motility patterns and velocity distributions. Thus the kinesin tail plays an active mechanical role beyond regulation or cargo binding, modulating microtubule behavior through inter-domain competition.

biophysics↗

Single depolymerizing and transport kinesins stabilize microtubule ends

Microtubules are highly dynamic cellular filaments and many intracellular processes like cell division depend on an accurate control of their length. Among other factors, microtubule length is actively modulated by motors from the kinesin superfamily. For example, yeast kinesin-8, Kip3, depolymerizes microtubules in a collective manner by a force- and length-dependent mechanism. However, whether single motors depolymerize or stabilize microtubule ends is unclear. Here, using interference reflection microscopy, we measured the influence of single kinesin motors on the stability of microtubules in an in vitro assay. Surprisingly, using unlabeled, stabilized microtubules, we found that both single kinesin-8 and non-depolymerizing kinesin-1 transport motors stabilized microtubule ends further by reducing the spontaneous microtubule depolymerization rate. Since we observed this effect for two very different kinesins, it implies a more general stabilization mechanism. For Kip3, this behavior is contrary to the collective force-dependent depolymerization activity of multiple motors. The complex, concentration-dependent interaction with microtubule ends provides new insights into the molecular mechanism of kinesin-8 and its regulatory function of microtubule length.

biophysics↗

Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

Observation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

biophysics↗

Germanium nanospheres for ultraresolution picotensiometry of kinesin motors

Kinesin motors are essential for transport of cellular cargo along cytoskeletal microtubule filaments. How motors step, detach, and cooperate is still unclear. To dissect the molecular motion of kinesin-1, we have developed germanium nanospheres as ultraresolution optical trapping probes. We found that single motors took 4-nm-center-of-mass steps. Furthermore, motors never detached from microtubules under native hindering load conditions. Instead, motors slid on microtubules with microsecond-long, 8-nm steps and remained in this slip state before detaching or reengaging in directed motion. Surprisingly, reengagement and, thus, rescue of directed motion was prevailing. We argue that teams of motors may be synchronized through this slip state and rescues need to be accounted for to understand long-range transport. One Sentence SummaryOptical trapping of high-refractive-index semiconductor nanoparticles shows how motors detach and walk with 4-nm steps.

biophysics↗

The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism

Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and length-dependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. Here, we tracked the microtubule end-binding activity of yeast kinesin-8, Kip3, under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors residence time at the microtubule end. On the microtubule lattice, loads also exponentially decreased the run length and time. However, for the same load, lattice run times were significantly longer compared to end residence times suggesting the presence of a distinct force-dependent detachment mechanism at the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism that unifies the so-called "bump-off" and "switching" models. Understanding the mechanics of kinesin-8s microtubule end activity will provide important insights into cell division with implications for cancer research. STATEMENT OF SIGNIFICANCEKinesin-8 motors are important for microtubule length regulation and are over-expressed in different types of cancer. Yet, on the molecular level, it is unclear how these motors depolymerize microtubules. Using high-precision optical tweezers, we measured how single yeast kinesin-8 motors, Kip3, interacted with the microtubule end. Interestingly, we found that single Kip3 motors were still motile at the microtubule end. The force dependence of how long single motors were associated with the microtubule end enabled us to estimate what force motors must exert onto each other to achieve the collective microtubule depolymerization speed of many motors. Our data support a collective force-dependent depolymerization mechanism. A better understanding of Kip3s microtubule end activity has implications for cell division and associated diseases.

biophysics↗