Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that 'curly', a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Whereas F-actin is semi-flexible with a persistence length of ~10 mm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 mm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures.
All data generated or analysed during the study are included in the manuscript and supporting files. Source data files for the actin curvature measurements (Figures 1-4) have been deposited and are freely availabe on Dyrad.
Calponin-Homology Domain mediated bending of membrane associated actin filamentsDryad Digital Repository, doi: 10.5061/dryad.bvq83bk6f.
- Mohan K Balasubramanian
- Mohan K Balasubramanian
- Darius Vasco Köster
- Saravanan Palani
- Scott Clarke
- Sayantika Ghosh
- Esther Ivorra-Molla
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Pekka Lappalainen, University of Helsinki, Finland
© 2021, Palani et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here, we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.
Swimming microorganisms often experience complex environments in their natural habitat. The same is true for microswimmers in envisioned biomedical applications. The simple aqueous conditions typically studied in the lab differ strongly from those found in these environments and often exclude the effects of small volume confinement or the influence that external fields have on their motion. In this work, we investigate magnetically steerable microswimmers, specifically magnetotactic bacteria, in strong spatial confinement and under the influence of an external magnetic field. We trap single cells in micrometer-sized microfluidic chambers and track and analyze their motion, which shows a variety of different trajectories, depending on the chamber size and the strength of the magnetic field. Combining these experimental observations with simulations using a variant of an active Brownian particle model, we explain the variety of trajectories by the interplay between the wall interactions and the magnetic torque. We also analyze the pronounced cell-to-cell heterogeneity, which makes single-cell tracking essential for an understanding of the motility patterns. In this way, our work establishes a basis for the analysis and prediction of microswimmer motility in more complex environments.