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

Abstract

Significance Complex 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.

Estimation of microtubule-generated forces using a DNA origami nanospring

“…This enabled us to measure forces generated by a protein complex tracking growing microtubule ends (Figure 3). Although the same phenomenon could also be observed using optical trapping (Alkemade et al, 2021; Molodtsov et al, 2016; Rodríguez-García et al, 2020), measurement of such small forces is usually technically challenging. It should be noted that nanosprings are less precise than optical tweezers in force estimation (uncertainty ~ 0.2 - 1.0 pN, Figure 1F, compared to ~ 0.1 pN or lower for optical tweezers).…”

“…Growing microtubule ends recruit end-binding (EB) proteins in the shape of a comet; these comets in turn recruit a number of secondary proteins that carry EB-binding SxIP motifs (Honnappa et al, 2009). The affinity of an EB comet to SxIP-containing proteins was reported to generate sub-pN forces that could extend membranes, transport actin filaments along with microtubule growth, and reverse the direction of a kinesin-14 motor (Alkemade et al, 2022; Molodtsov et al, 2016; Rodríguez-García et al, 2020). While prior measurements were performed using optical trapping, measuring sub-pN forces using this method is challenging, because it is easy to lose a bead from a soft trap.…”

Actin Architecture Steers Microtubules in Active Cytoskeletal Composite

“…The recognition of actin architecture by microtubules in mammalian cell cortex is governed by the microtubule dynamics, 48,49 cross-linking 50,51 and retrograde actin flow, 52−54 which, in contrast to our assay, result in negligible movement of microtubule lattice relative to actin network. 55,56 Nevertheless, our findings highlight the importance of the effects that nonspecific steric interactions may have in biological or bioinspired systems.…”

Visualizing Actin and Microtubule Coupling Dynamics <em>In Vitro</em> by Total Internal Reflection Fluorescence (TIRF) Microscopy

“… Measure microtubule growth speeds from kymograph slopes ( Figure 3A , 1–2; slope of black lines). Count dynamic microtubule events (catastrophe or regrowth) from the generated kymograph or using available analysis macros 5 , 8 , 18 , 25 . Red dotted lines in Figure 3A , 1–2 represent catastrophe/rapid disassembly events.…”

“…Growing evidence suggests this single polymer approach has concealed the dual functions of some of the proteins/complexes that enable actin-microtubule coupling events 7 , 8 , 9 , 10 , 11 , 12 , 13 . Experiments where both polymers are present are rare and often define mechanisms with a single dynamic polymer and static stabilized version of the other 6 , 8 , 9 , 10 , 11 , 14 , 15 , 16 , 17 , 18 . Thus, methods are needed to investigate the emergent properties of actin-microtubule coordinating proteins that may only be fully understood in experimental systems that employ both dynamic polymers.…”