The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule Sliding

Ignas Gaska, Mason E. Armstrong, April Alfieri, Scott Forth

Cell division in eukaryotes requires the regulated assembly of the spindle apparatus. The proper organization of microtubules within the spindle is driven by motor proteins that exert forces to slide filaments, whereas non-motor proteins crosslink filaments into higher-order motifs, such as overlapping bundles. It is not clear how active and passive forces are integrated to produce regulated mechanical outputs within spindles. Here, we employ simultaneous optical trapping and total internal reflection fluorescence (TIRF) microscopy to directly measure the frictional forces produced by the mitotic crosslinking protein PRC1 that resist microtubule sliding. These forces scale with microtubule sliding velocity and the number of PRC1 crosslinks but do not depend on overlap length or PRC1 density within overlaps. Our results suggest that PRC1 ensembles act similarly to a mechanical dashpot, producing significant resistance against fast motions but minimal resistance against slow motions, allowing for the integration of diverse motor activities into a single mechanical outcome.

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Probing the Functional Role of Physical Motion in Development

Moritz Kreysing

Spatiotemporal organization during development has frequently been proposed to be explainable by reaction-transport models, where biochemical reactions couple to physical motion. However, whereas genetic tools allow causality of molecular players to be dissected via perturbation experiments, the functional role of physical transport processes, such as diffusion and cytoplasmic streaming, frequently remains untestable. This Perspective explores the challenges of validating reaction-transport hypotheses and highlights new opportunities provided by perturbation approaches that specifically target physical transport mechanisms. Using these methods, experimental physics may begin to catch up with molecular biology and find ways to test roles of diffusion and flows in development.

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Mechanical Force-Driven Adherens Junction Remodeling and Epithelial Dynamics

Diana Pinheiro, Yohanns Bellaïche

During epithelial tissue development, repair, and homeostasis, adherens junctions (AJs) ensure intercellular adhesion and tissue integrity while allowing for cell and tissue dynamics. Mechanical forces play critical roles in AJs’ composition and dynamics. Recent findings highlight that beyond a well-established role in reinforcing cell-cell adhesion, AJ mechanosensitivity promotes junctional remodeling and polarization, thereby regulating critical processes such as cell intercalation, division, and collective migration. Here, we provide an integrated view of mechanosensing mechanisms that regulate cell-cell contact composition, geometry, and integrity under tension and highlight pivotal roles for mechanosensitive AJ remodeling in preserving epithelial integrity and sustaining tissue dynamics.

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Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules

Yuta Shimamoto, Scott Forth, Tarun M. Kapoor

The proper organization of the microtubule-based mitotic spindle is proposed to depend on nanometer-sized motor proteins generating forces that scale with a micron-sized geometric feature, such as microtubule overlap length. However, it is unclear whether such regulation can be achieved by any mitotic motor protein. Here, we employ an optical-trap- and total internal reflection fluorescence (TIRF)-based assay to show that ensembles of kinesin-5, a conserved mitotic motor protein, can push apart overlapping antiparallel microtubules to generate a force whose magnitude scales with filament overlap length. We also find that kinesin-5 can produce overlap-length-dependent “brake-like” resistance against relative microtubule sliding in both parallel and antiparallel geometries, an activity that has been suggested by cell biological studies but had not been directly measured. Together, these findings, along with numerical simulations, reveal how a motor protein can function as an analog converter, “reading” simple geometric and dynamic features in cytoskeletal networks to produce regulated force outputs.

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Actin Filament Turnover Drives Leading Edge Growth during Myelin Sheath Formation in the Central Nervous System

Schanila Nawaz, Paula Sánchez, Sebastian Schmitt, Nicolas Snaidero, Mišo Mitkovski, Caroline Velte, Bastian R. Brückner, Ioannis Alexopoulos, Tim Czopka, Sang Y. Jung, Jeong S. Rhee, Andreas Janshoff, Walter Witke, Iwan A.T. Schaap, David A. Lyons, Mikael Simons

During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.

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Membrane Shape Modulates Transmembrane Protein Distribution

Sophie Aimon, Andrew Callan-Jones, Alice Berthaud, Mathieu Pinot, Gilman E.S. Toombes, Patricia Bassereau

Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins.

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Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors

Mary Williard Elting, James A. Spudich

Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.
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Optical Tweezers Studies on Notch: Single-Molecule Interaction Strength Is Independent of Ligand Endocytosis

Bhupinder Shergill, Laurence Meloty-Kapella, Abdiwahab A. Musse, Gerry Weinmaster, Elliot Botvinick

Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force. 

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Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin

Laurence Meloty-Kapella, Bhupinder Shergill, Jane Kuon, Elliot Botvinick, Gerry Weinmaster

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.

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Mechanistic Basis of Otolith Formation during Teleost Inner Ear Development

David Wu, Jonathan B. Freund, Scott E. Fraser and Julien Vermot

Otoliths, which are connected to stereociliary bundles in the inner ear, serve as inertial sensors for balance. In teleostei, otolith development is critically dependent on flow forces generated by beating cilia; however, the mechanism by which flow controls otolith formation remains unclear. Here, we have developed a noninvasive flow probe using optical tweezers and a viscous flow model in order to demonstrate how the observed hydrodynamics influence otolith assembly. We show that rotational flow stirs and suppresses precursor agglomeration in the core of the cilia-driven vortex. The velocity field correlates with the shape of the otolith and we provide evidence that hydrodynamics is actively involved in controlling otolith morphogenesis. An implication of this hydrodynamic effect is that otolith self-assembly is mediated by the balance between Brownian motion and cilia-driven flow. More generally, this flow feature highlights an alternative biological strategy for controlling particle localization in solution.

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