Abstract
Over the last 25 years, extensive progress has been made in developing a range of nanotechnological applications where cytoskeletal filaments and molecular motors are key elements. This ...includes novel, highly miniaturized lab on a chip systems for biosensing, nanoseparation etc but also new materials and parallel computation devices for solving otherwise intractable mathematical problems. For such approaches, both actin-based and microtubule-based cytoskeletal systems have been used. However, in accordance with their different cellular functions, actin filaments and microtubules have different properties and interaction kinetics with molecular motors. Therefore, the two systems obviously exhibit different advantages and encounter different challenges when exploited for applications. Specifically, the achievable filament velocities, the capability to guide filaments along nanopatterned tracks and the capability to attach and transport cargo differ between actin- and microtubule-based systems. Our aim here is to systematically elucidate these differences to facilitate design of new devices and optimize future developments. We first review the cellular functions and the fundamental physical and biochemical properties of actin filaments and microtubules. In this context we also consider their interaction with molecular motors and other regulatory proteins that are of relevance for applications. We then relate these properties to the advantages and challenges associated with the use of each of the motor-filament systems for different tasks. Finally, fundamental properties are considered in relation to some of the most interesting future development paths e.g. in biosensing and biocomputation.
Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with frequent directional reversals ...owing to the simultaneous presence of opposite-polarity motors. So far, it has been unclear whether such complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires regulators. Here, we demonstrate that a minimal system, comprising purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) attached to large unilamellar vesicles, faithfully reproduces in vivo cargo motility, including runs, pauses, and reversals. Remarkably, opposing motors do not affect vesicle velocity during runs. Our computational model reveals that the engagement of a small number of motors is pivotal for transitioning between runs and pauses. Taken together, our results suggest that motors bound to vesicular cargo transiently engage in a tug-of-war during pauses. Subsequently, stochastic motor attachment and detachment events can lead to directional reversals without the need for regulators.
Obstacles on the surface of microtubules can lead to defective cargo transport, proposed to play a role in neurological diseases such as Alzheimer’s. However, little is known about how motor ...proteins, which follow individual microtubule protofilaments (such as kinesin-1), deal with obstacles on the molecular level. Here, we used rigor-binding mutants of kinesin-1 as roadblocks to permanently obstruct individual microtubule binding sites and studied the movement of individual kinesin-1 motors by single-molecule fluorescence and dark-field scattering microscopy in vitro. In the presence of roadblocks, kinesin-1 often stopped for ∼0.4 s before either detaching or continuing to move, whereby the latter circumvention events occurred in >30% after a stopping event. Consequently, and in agreement with numerical simulations, the mean velocity, mean run length, and mean dwell time of the kinesin-1 motors decreased upon increasing the roadblock density. Tracking individual kinesin-1 motors labeled by 40 nm gold particles with 6 nm spatial and 1 ms temporal precision revealed that ∼70% of the circumvention events were associated with significant transverse shifts perpendicular to the axis of the microtubule. These side-shifts, which occurred with equal likelihood to the left and right, were accompanied by a range of longitudinal shifts suggesting that roadblock circumvention involves the unbinding and rebinding of the motors. Thus, processive motors, which commonly follow individual protofilaments in the absence of obstacles, appear to possess intrinsic circumvention mechanisms. These mechanisms were potentially optimized by evolution for the motor’s specific intracellular tasks and environments.
The cytoskeleton is a network of interconnected protein filaments, which provide a three‐dimensional scaffold for cells. Remodeling of the cytoskeleton is important for key cellular processes, such ...as cell motility, division, or morphogenesis. This remodeling is traditionally considered to be driven exclusively by processes consuming chemical energy, such as the dynamics of the filaments or the action of molecular motors. Here, we review two mechanisms of cytoskeletal network remodeling that are independent of the consumption of chemical energy. In both cases directed motion of overlapping filaments is driven by entropic forces, which arise from harnessing thermal energy present in solution. Entropic forces are induced either by macromolecular crowding agents or by diffusible crosslinkers confined to the regions where filaments overlap. Both mechanisms increase filament overlap length and lead to the contraction of filament networks. These force‐generating mechanisms, together with the chemical energy‐dependent mechanisms, need to be considered for the comprehensive quantitative picture of the remodeling of cytoskeletal networks in cells.
Directional sliding of overlapping filaments relative to each other is one of the key means of the remodeling of cytoskeletal filament networks. Traditionally, filament sliding is considered to be driven by molecular motors. Recent findings show that directional filament sliding can be also driven by macromolecular crowding or by non‐enzymatic diffusible filament crosslinkers confined in the filament overlaps.
Engineering cargo-loading strategies is crucial to developing nanotechnological applications of microtubule-based biomolecular transport systems. Here, we report a highly efficient and robust ...bioconjugation scheme to load antibodies to microtubules. Our method takes advantage of the inverse-electron-demand Diels–Alder addition reaction between tetrazine and trans-cyclooctene: the fastest known bioorthogonal reaction, characterized by its excellent selectivity and biocompatibility. As proof of concept, we performed kinesin-1 gliding motility assays with antibody-conjugated microtubules and demonstrated the highly sensitive detection of fluorescent protein analyte down to 0.1 pM in microliter sample volumes. Importantly, the detection selectivity was retained in the presence of other fluorescent background proteins. We envision the applicability of our fast, simple, and robust conjugation method to a wide range of biosensing platforms based on biomolecular transport systems.
Constriction of the cytokinetic ring, a circular structure of actin filaments, is an essential step during cell division. Mechanical forces driving the constriction are attributed to myosin motor ...proteins, which slide actin filaments along each other. However, in multiple organisms, ring constriction has been reported to be myosin independent. How actin rings constrict in the absence of motor activity remains unclear. Here, we demonstrate that anillin, a non-motor actin crosslinker, indispensable during cytokinesis, autonomously propels the contractility of actin bundles. Anillin generates contractile forces of tens of pico-Newtons to maximise the lengths of overlaps between bundled actin filaments. The contractility is enhanced by actin disassembly. When multiple actin filaments are arranged into a ring, this contractility leads to ring constriction. Our results indicate that passive actin crosslinkers can substitute for the activity of molecular motors to generate contractile forces in a variety of actin networks, including the cytokinetic ring.
Within the mitotic spindle, kinesin motors cross-link and slide overlapping microtubules. Some of these motors exhibit off-axis power strokes, but their impact on motility and force generation in ...microtubule overlaps has not been investigated. Here, we develop and utilize a three-dimensional in vitro motility assay to explore kinesin-14, Ncd, driven sliding of cross-linked microtubules. We observe that free microtubules, sliding on suspended microtubules, not only rotate around their own axis but also move around the suspended microtubules with right-handed helical trajectories. Importantly, the associated torque is large enough to cause microtubule twisting and coiling. Further, our technique allows us to measure the in situ spatial extension of the motors between cross-linked microtubules to be about 20 nm. We argue that the capability of microtubule-crosslinking kinesins to cause helical motion of overlapping microtubules around each other allows for flexible filament organization, roadblock circumvention and torque generation in the mitotic spindle.
We analyze the brane content and charges in all of the orientifold string theories on space-times of the form
E
×
R
8
, where
E
is an elliptic curve with holomorphic or anti-holomorphic involution. ...Many of these theories involve “twistings” coming from the
B
-field and/or sign choices on the orientifold planes. A description of these theories from the point of view of algebraic geometry, using the Legendre normal form, naturally divides them into three groupings. The physical theories within each grouping are related to one another via sequences of
T
-dualities. Our approach agrees with both previous topological calculations of twisted
KR
-theory and known physics arguments, and explains how the twistings originate from both a mathematical and a physical perspective.
Owing to their wide spectrum of in vivo functions, motor proteins, such as kinesin-1, show great potential for application as nanomachines in engineered environments. When attached to a substrate ...surface, these motors are envisioned to shuttle cargo that is bound to reconstituted microtubules--one component of the cell cytoskeleton--from one location to another. One potentially serious problem for such applications is, however, the rotation of the microtubules around their longitudinal axis. Here we explore this issue by labelling the gliding microtubules with quantum dots to simultaneously follow their sinusoidal side-to-side and up-and-down motion in three dimensions with nanometre accuracy. Microtubule rotation, which originates from the kinesin moving along the individual protofilaments of the microtubule, was not impeded by the quantum dots. However, pick-up of large cargo inhibited the rotation but did not affect the velocity of microtubule gliding. Our data show that kinesin-driven microtubules make flexible, responsive and effective molecular shuttles for nanotransport applications.
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