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Alkemade, C.

Publications and source records attributed to Alkemade, C..

3 recordsLinked to original sources

Cross-linkers at growing microtubule ends generate forces that drive actin transport

The actin and microtubule cytoskeletons form active networks in the cell that can contract and remodel, resulting in vital cellular processes as cell division and motility. Motor proteins play an important role in generating the forces required for these processes, but more recently the concept of passive cross-linkers being able to generate forces has emerged. So far, these passive cross-linkers have been studied in the context of separate actin and microtubule systems. Here, we show that crosslinkers also allow actin and microtubules to exert forces on each other. More specifically, we study single actin filaments that are cross-linked to growing microtubule ends, using in vitro reconstitution, computer simulations, and a minimal theoretical model. We show that microtubules can transport actin filaments over large (micrometer-range) distances, and find that this transport results from two antagonistic forces arising from the binding of cross-linkers to the overlap between the actin and microtubule filaments. The cross-linkers attempt to maximize the overlap between the actin and the tip of the growing microtubules, creating an affinity-driven forward condensation force, and simultaneously create a competing friction force along the microtubule lattice. We predict and verify experimentally how the average transport time depends on the actin filament length and the microtubule growth velocity, confirming the competition between a forward condensation force and a backward friction force. In addition, we theoretically predict and experimentally verify that the condensation force is of the order of 0.1 pN. Thus, our results reveal a new mechanism for local actin remodeling by growing microtubules. Significance StatementComplex cellular processes such as cell migration require coordinated remodeling of both the actin and the microtubule cytoskeleton. The two networks for instance exert forces on each other via active motor proteins. Here we show that, surprisingly, coupling via passive cross-linkers can also result in force generation. We specifically study the transport of actin filaments by growing microtubule ends. We show by cell-free reconstitution experiments, computer simulations, and theoretical modeling that this transport is driven by the affinity of the cross-linker for the chemically distinct microtubule tip region. Our work predicts that growing microtubules could potentially rapidly relocate newly nucleated actin filaments to the leading edge of the cell and thus boost migration.

biophysics↗

Revealing the Assembly of Filamentous Proteins with Scanning Transmission Electron Microscopy

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments.

biophysics↗

Frustrated binding of biopolymer crosslinkers

Transiently crosslinked actin filament networks allow cells to combine elastic rigidity with the ability to deform viscoelastically. Theoretical models of semiflexible polymer networks predict that the crosslinker unbinding rate governs the timescale beyond which viscoelastic flow occurs. However a direct comparison between network and crosslinker dynamics is lacking. Here we measure the networks stress relaxation timescale using rheology and the lifetime of bound crosslinkers using fluorescence recovery after photobleaching. lntruigingly, we observe that the crosslinker unbinding rate measured by FRAP is more than an order of magnitude slower than the rate measured by rheology. We rationalize this difference with a three-state model where crosslinkers are bound to either 0, 1 or 2 filaments, which allows us to extract crosslinker transition rates that are otherwise difficult to access. We find that the unbinding rate of singly bound crosslinkers is nearly two orders of magnitude slower than for double bound ones. We attribute the increased unbinding rate of doubly bound crosslinkers to the high stiffness of biopolymers, which frustrates crosslinker binding.

biophysics↗