Biology subjects

Ruhnow, F.

Publications and source records attributed to Ruhnow, F..

5 recordsLinked to original sources

bioRxiv2024

IQD2 recruits KLCR1 to the membrane-microtubule nexus to promote cytoskeletal mechano-responsiveness in leaf epidermis pavement cells

Plant cells experience a variety of mechanical stresses from both internal and external sources, including turgor pressure, mechanical strains arising from heterogeneous growth between neighboring cells, and environmental factors like touch from soil, rain, or wind [1,2]. These stresses serve as signals at the cell-, tissue- and organismal level to coordinate plant growth during development and stress responses [3]. In plants, the physical cell wall-plasma membrane-microtubule continuum is proposed to be integral in transducing mechanical signals from the exterior to intracellular components [4-6]. Cortical microtubules (CMTs) rapidly reorient in response to mechanical stress to align with the maximal tensile stress direction [7,8]. Several studies proposed that CMTs themselves may act as stress sensors; the precise mechanisms involved in the regulation of CMTs and the modes of sensing, however, are still not clearly understood. Here, we show that IQD2 and KLCR1 are enriched at CMTs in proximity to the plasma membrane. IQD2, which is a bona fide microtubule-associated protein, promotes microtubule localization of KLCR1. By combining cross-linking mass spectrometry (XL-MS) and computational modeling with structure-function studies, we present first experimental insights into the composition and structure of IQD2-KLCR1 complexes. Further, we demonstrate that the IQD2-KLCR1 module is a positive regulator of microtubule mechano-responses in pavement cells. Collectively, our work identifies the IQD2-KLCR1 module as novel regulator of mechanostress-mediated CMT reorientation and provides a framework for future mechanistic studies aimed at a functional dissection of mechanotransduction at the plasma membrane-CMT interface during growth and plant morphogenesis. HighlightsO_LIIQD2 and KLCR1 localize to the plasma membrane-microtubule nexus C_LIO_LIIQD2 is required for efficient microtubule targeting of KLCR1 in planta C_LIO_LIIQD2 physically interacts with KLCR1 and microtubules C_LIO_LIThe IQD2-KLCR1 module promotes mechano-stress induced microtubule reorganization C_LI

plant biology↗

bioRxiv2024

NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

Nuclear mitotic apparatus protein (NuMA) is indispensable for the mitotic functions of the major microtubule minus-end directed motor cytoplasmic dynein 1. NuMA and dynein are both essential for correct spindle pole organization. How these proteins cooperate to gather microtubule minus ends at spindle poles remains unclear. Here we use microscopy-based in vitro reconstitutions to demonstrate that NuMA is a dynein adaptor, activating processive dynein motility together with dyneins cofactors dynactin and Lissencephaly-1 (Lis1). Additionally, we find that NuMA binds and stabilizes microtubule minus ends, allowing dynein/dynactin/NuMA to transport microtubule minus ends as cargo to other minus ends. We further show that the microtubule-nucleating {gamma}-tubulin ring complex ({gamma}TuRC) hinders NuMA binding. This shows that either {gamma}TuRC needs to be released or microtubules need to be severed to generate free minus ends before they can be incorporated into spindle poles by dynein/dynactin/NuMA. These results provide new mechanistic insight into how dynein, dynactin, NuMA and Lis1 cooperate to organize spindle poles in cells.

biochemistry↗

bioRxiv2023

Microtubule binding of the human HAUS complex is directly controlled by importins and Ran-GTP

Mitotic spindle assembly during cell division is a highly regulated process. Ran-GTP produced around chromosomes controls the activity of a multitude of spindle assembly factors by releasing them from inhibitory interaction with importins. A major consequence of Ran-GTP regulation is the stimulation of local microtubule nucleation around chromosomes via augmin/HAUS-mediated branched microtubule nucleation, a process that is critically important for correct spindle assembly. However, augmin is not known to be a direct target of the Ran-GTP pathway, raising the question of how its activity is controlled. Here we present the in vitro reconstitution of Ran-GTP-regulated microtubule binding of the human HAUS complex. We demonstrate that importins directly bind to the HAUS complex, which prevents HAUS from binding to microtubules. Ran-GTP relieves this inhibition. Therefore, the HAUS complex is a direct target of the Ran-GTP pathway, suggesting that branching microtubule nucleation is directly regulated by the Ran-GTP gradient around chromosomes in dividing cells.

cell biology↗

bioRxiv2019

The kinesin-14, Ncd, drives a right-handed, helical motion of antiparallel microtubules around each other

Within the mitotic spindle, several kinesin motors crosslink and slide microtubules. While some of them (e.g. kinesin-5, kinesin-8 and kinesin-14) have been shown to exhibit sideways components in their step cycles, the impact of the resulting off-axis power strokes on motility and force generation in the spindle has not been investigated so far. Here, we develop and utilize a novel three-dimensional in vitro motility assay to explore the kinesin-14, Ncd, driven sliding of crosslinked, fluorescently-labeled microtubules. We find that free microtubules, sliding in an antiparallel orientation on microtubules suspended between nanofabricated ridges, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. In contrast, microtubules crosslinked in parallel orientation are static with neither longitudinal nor helical motion. Further, our technique allows us to measure the in situ spatial extension of the motors between the crosslinked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.

biophysics↗

bioRxiv2017

Challenges in estimating the motility parameters of single processive motor proteins

Cytoskeletal motor proteins are essential to the function of a wide range of intracellular mechanosystems. The biophysical characterization of their movement along their filamentous tracks is therefore of large importance. Towards this end, single-molecule, in vitro stepping-motility assays are commonly used to determine motor velocity and run length. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental conditions and the data analysis methods. Here, we investigate the movement of fluorescently-labeled, processive, dimeric motor proteins and propose a unified algorithm to correct the measurements for finite filament length as well as photobleaching. Particular emphasis is put on estimating the statistical errors associated with the proposed evaluation method as knowledge of these values is crucial when comparing measurements from different experiments. Testing our approach with simulated and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules, we show (i) that velocity distributions should be fitted by a t location-scale probability density function rather than by a norm*al distribution, (ii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iii) that the interaction time and run length of the motors can be estimated independent of the filament length distribution, and (iv) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. Moreover, our analysis reveals that controlling the temperature during the experiments with a precision below 1 K is of importance. We believe, our method will not only improve the evaluation of experimental data, but will also allow for better statistical comparisons between different populations of motor proteins (e.g. with distinct mutations or linked to different cargos) and filaments (e.g. in distinct nucleotide states or with different posttranslational modifications).

biophysics↗