The dynein drives these small clusters together, fusing them to create larger and larger networks. As the motor protein continues to jam the microtubules together, the network contracts, until it can't get any smaller. Based on this experiment, the researchers developed a model that quantifies and describes this behavior and lends insight into not only spindle assembly but also self-organization in general.
This model could provide insights into how to design materials that can self-assemble or autonomously contract, like a self-squeezing sponge. Original written by Leah Burrows. Note: Content may be edited for style and length. Science News. The research was recently published in the journal eLife.
Active contraction of microtubule networks. ScienceDaily, 28 January Harvard School of Engineering and Applied Sciences. Cell division: Microtubules, assemble!. Intermediate filaments commonly work in tandem with microtubules, providing strength and support for the fragile tubulin structures. All cells have intermediate filaments, but the protein subunits of these structures vary.
Some cells have multiple types of intermediate filaments, and some intermediate filaments are associated with specific cell types. For example, neurofilaments are found specifically in neurons most prominently in the long axons of these cells , desmin filaments are found specifically in muscle cells, and keratins are found specifically in epithelial cells. Other intermediate filaments are distributed more widely.
For example, vimentin filaments are found in a broad range of cell types and frequently colocalize with microtubules. Similarly, lamins are found in all cell types, where they form a meshwork that reinforces the inside of the nuclear membrane. Note that intermediate filaments are not polar in the way that actin or tubulin are Figure 4. Figure 4: The structure of intermediate filaments Intermediate filaments are composed of smaller strands in the shape of rods. Eight rods are aligned in a staggered array with another eight rods, and these components all twist together to form the rope-like conformation of an intermediate filament.
Cytoskeletal filaments provide the basis for cell movement. For instance, cilia and eukaryotic flagella move as a result of microtubules sliding along each other. In fact, cross sections of these tail-like cellular extensions show organized arrays of microtubules.
Other cell movements, such as the pinching off of the cell membrane in the final step of cell division also known as cytokinesis are produced by the contractile capacity of actin filament networks. Actin filaments are extremely dynamic and can rapidly form and disassemble.
In fact, this dynamic action underlies the crawling behavior of cells such as amoebae. At the leading edge of a moving cell, actin filaments are rapidly polymerizing; at its rear edge, they are quickly depolymerizing Figure 5.
A large number of other proteins participate in actin assembly and disassembly as well. Figure 5: Cell migration is dependent on different actin filament structures. These protrusive structures contain actin filaments, with elongating barbed ends orientated toward the plasma membrane. B During cellular arm extension, the plasma membrane sticks to the surface at the leading edge. C Next, the nucleus and the cell body are pushed forward through intracellular contraction forces mediated by stress fibers.
D Then, retraction fibers pull the rear of the cell forward. Filopodia: molecular architecture and cellular functions. Nature Reviews Molecular Cell Biology 9, All rights reserved. Figure Detail. This page appears in the following eBook. Aa Aa Aa. Microtubules and Filaments. What Is the Cytoskeleton Made Of? The cytoskeleton of eukaryotic cells is made of filamentous proteins, and it provides mechanical support to the cell and its cytoplasmic constituents.
All cytoskeletons consist of three major classes of elements that differ in size and in protein composition. Microtubules are the largest type of filament, with a diameter of about 25 nanometers nm , and they are composed of a protein called tubulin. Actin filaments are the smallest type, with a diameter of only about 6 nm, and they are made of a protein called actin.
Even for simple materials, mechanical responses can depend on whether forces are applied quickly or slowly, as illustrated by the traditional classroom demonstration using a mixture of cornstarch and water. This viscoelastic material behaves as a solid when rapid stirring is attempted, but adopts a more liquid-like behavior when stirring forces are applied slowly.
At the molecular level, individual motor proteins are capable of moving at rates ranging from tens to hundreds of nanometers per second Sharp et al. Within cells, microtubule plus ends grow at rates on the order of several hundred nanometers per second Rusan et al.
Therefore, both motor proteins and microtubule dynamics can exert forces rapidly, with motor stepping and tubulin addition events typically occurring many of times per second at the nanometer scale. Frictional forces are similarly likely to arise at similar fast time scales, as proteins interact with sliding microtubules or dynamic filament ends.
Elastic forces, such as k-fiber bending or kinetochore stretching across paired chromosomes, are more likely to persist on the order of minutes during metaphase. Therefore, when formulating a spindle force map, one must account for not only active and passive forces generated by different individual components within the spindle, but also the timescale on which these forces act. How might we begin to understand the micromechanics of the metaphase spindle?
Spindles assembled in meiotic extract from Xenopus laevis eggs have proven to be an especially powerful system to begin to address this question, as these spindles are not constrained within cellular membranes, and therefore direct access to the spindle can be achieved with both chemical and mechanical probes. Using calibrated microneedles, the time scale—dependent viscoelastic properties of the metaphase spindle have been directly examined Shimamoto et al. In brief, two glass needles were first coated with silicone to suppress specific binding interactions with individual spindle components Fig.
The needles were inserted into the spindle near the metaphase plate, and perturbations were generated perpendicular to the long axis of the spindle by oscillating one needle, while the second served as a force-calibrated readout.
At slow minutes and fast seconds time scales, the spindle responded as an elastic material, stretching in response to micrometer-scale motions. At intermediate time scales tens of seconds , the spindle exhibited viscous properties, behaving more like a liquid. These time scale—dependent mechanical properties can be well described as a Zener-type viscoelastic solid Fig. This model consists of two springs and a dashpot. One of the spring-like elements, in which a stretching force produces a specific amount of displacement, may be linked to the bending stiffness of interpolar microtubules.
In series with this spring is a dashpot element that acts as a viscous frictional damper that resists motion and may be linked to the lifetimes and strengths of microtubule cross-links.
These two elements together are then in parallel with a stiffer spring-like element that likely corresponds to k-fiber bending. The relative contributions of spindle components were determined by biochemical disruption of specific populations of microtubules or mitotic proteins and direct mechanical measurement of the perturbed spindle. Applying forces directly at the spindle poles and stretching outward along the long axis reveals that the spindle is able to generate a restoring force that persists for many minutes Takagi et al.
During this stretching, spindle volume and tubulin density were conserved, as determined by fluorescence microscopy Fig. These experiments revealed the time-dependent viscoelastic properties of the spindle microtubule network, with dynamic interpolar microtubules, long-lived and stiff kinetochore fibers, and protein-mediated microtubule cross-linking, all contributing to the organization of a robust network that can remodel itself to accommodate chromosome motions.
Recent biophysical studies have begun to elucidate new properties that describe these spindle mechanics and help to refine the map of forces across the spindle network. The use of reconstituted microtubule networks by small ensembles of purified proteins has proven useful in measuring the mechanics of microtubule network assembly.
From these studies, new principles and concepts about force production are beginning to emerge. Kinesin-5 was the first motor protein shown to slide microtubules apart by walking on each filament it cross-links Kapitein et al. Kinesin-5 functions as a homotetramer and adopts a bipolar structure, with pairs of N terminus motor domains located at opposite ends of a coiled-coil tetramerization domain Kashina et al.
Processive directional motion is triggered by cross-linking, and microtubule sliding is enhanced by a nonmotor microtubule-binding domain at the C terminus Weinger et al. This ability to push apart antiparallel microtubules is linked to the various cellular functions of kinesin-5, which include establishing spindle bipolarity and maintenance, as well as contributing to the poleward flux of microtubules in metaphase Ferenz et al. The sliding of microtubule pairs has also been demonstrated for two minus end motor proteins.
Kinesin Ncd is a microtubule minus end—directed kinesin, and it contains a C terminus motor domain and an N terminus nonmotor microtubule-binding domain. These domains also allow kinesin to bundle and slide antiparallel microtubules Fink et al.
Furthermore, the yeast kinesin homologue Klp2 has been shown to assemble bundles of parallel microtubules Braun et al. Another important minus end—directed motor protein, cytoplasmic dynein, has been shown to slide apart two microtubules in a reconstituted microtubule bundle assay Tanenbaum et al. It has been assumed that forces are generated as a result of these motor proteins driving relative microtubule sliding, but this was not directly measured until recent experimental tests using kinesin-5 Shimamoto et al.
Traditionally, in single-molecule assays, individual proteins of interest were directly conjugated to the trapping bead, which could then be brought into contact with the microtubule substrate. However, to measure the force generated within sliding microtubule pairs cross-linked by an ensemble of kinesin-5 molecules, it would be advantageous to attach the bead directly to one of the microtubules while the second microtubule is immobilized on a surface.
Additionally, a method for determining the number of kinesin-5 molecules in the ensemble and the length of overlap between the two microtubules would be required. These criteria have recently been met, and the force production by kinesin-5 ensembles acting within cross-linked microtubule pairs has now been directly monitored, revealing that the magnitudes of both pushing and braking forces scale in proportion to the length of microtubule overlap Shimamoto et al.
Previous analyses of cargos carried by multiple kinesin-1 molecules suggest that the force may not persistently scale as more motor proteins engage the microtubule surface Jamison et al. Indeed, cross-talk between two or more kinesin molecules may lead to the rapid detachment of one or more of the proteins as the stepping behavior of one kinesin interferes with the mechanics of adjacent kinesins Furuta et al.
Within these types of systems, if one motor protein steps, a strain is induced along the protein that propagates to other proteins in the ensemble. If the microtubule binding of these other molecules is strongly sensitive to force, this strain will accelerate the unbinding of motor proteins, and therefore persistent force cannot build up. In contrast, during the sliding of antiparallel microtubules by kinesin-5, the magnitude of the sliding force that can be generated scales with both the length of the overlap between antiparallel microtubules and the number of motor proteins that are engaged in cross-linking Shimamoto et al.
This result suggests that kinesin-5 molecules do not interfere with, but rather mainly slow down, the stepping of adjacent molecules in a force-dependent manner without inducing microtubule unbinding or loss of cross-linking.
Moreover, it was shown that when the microtubules are moving at velocities faster than the intrinsic kinesin-5 stepping rate, an ensemble of kinesin-5 proteins generates a resistive braking force, which, remarkably, also scales with the length of microtubule overlap Shimamoto et al. Parallel microtubule overlaps cross-linked by ensembles of kinesin-5 were also examined Shimamoto et al.
Interestingly, the magnitude of these force fluctuations did not depend on overlap length. However, length- and velocity-dependent braking forces against microtubule sliding were measured for parallel overlaps. Taking together the observations for both antiparallel and parallel cross-linking geometries, we concluded that kinesin-5 ensembles can serve as a regulator of microtubule sliding velocity within the spindle, producing larger forces when antiparallel overlaps are long, smaller forces when antiparallel overlaps are short, and braking forces against the relative sliding of antiparallel microtubules moving faster than the natural rate of kinesin-5 stepping and parallel microtubules moving across a range of velocities measured.
Motor proteins with opposing directional preferences can occupy the same overlapping region within microtubule bundles in spindles Fig. In such situations, an antagonistic behavior can emerge; for example, if one class of protein slides plus ends of microtubules apart whereas the other slides minus ends apart.
Examination of microtubule bundles cross-linked by both kinesin-5 and kinesin in vitro revealed that a stable force balance within this minimal system could not be achieved Fig. At similar fractional amounts of the two proteins, an unstable fluctuation microtubule motion was observed; titrating more of one motor protein versus the other resulted in directional motion Hentrich and Surrey, The addition of an artificial microtubule cross-linking construct that lacked motor activity served to stabilize this system and significantly reduced the magnitude of unstable fluctuations.
This combination of active force generators and passive brake-like elements acting within the same microtubule network could produce stable overlaps that persisted for minutes.
In light of these observations, it is clear that a careful characterization is needed of passive microtubule cross-linkers under controlled loads, at different timescales, and with consideration of microtubule orientation. Inspired by studies of molecular friction generated by the kinesin-8 motor protein Kip3 Bormuth et al.
Examined in these assays were NuMA, a large protein that bundles microtubules and localizes to the metaphase spindle pole; EB1, a plus end—binding protein that tracks the growing tips of microtubules; and PRC1, a homodimeric protein in the MAP65 family that preferentially cross-links antiparallel microtubules while localizing to the spindle midzone in anaphase. We demonstrated that these proteins all produce frictional resistance whose magnitude depends nonlinearly on the speed at which the protein was dragged across the microtubule lattice Fig.
Additionally, differences in the microtubule binding structural motifs for each of the proteins examined likely resulted in different strengths of frictional interactions. Might it be possible for a single molecule to generate enough friction to modulate the speed of a motor protein? Although this may suggest that a single MAP—microtubule interaction would not provide substantial resistance against motor protein—generated forces, it could be the case that clustered ensembles of such molecules would result in a scaling of the total resistive force.
For example, a cluster of only 10 MAPs interacting with a single microtubule could provide piconewtons of resistive load, which would be enough to reduce or even stall the stepping velocity of a motor protein. Indeed, the yeast MAP65, Ase1, can prevent sliding microtubules that are driven by kinesin from completely falling apart Braun et al.
These results suggest that proteins that bind microtubules may have the ability to act as brakes, resisting the motions of filaments within a dense network such as the spindle. Interestingly, diverse frictional asymmetries were also observed across these studies Fig. For example, both Kip3 and EB1 produced less frictional resistance when dragged toward microtubule plus ends Bormuth et al.
In contrast, NuMA produced less frictional resistance when moving toward minus ends, consistent with its strong localization at spindle poles where minus ends predominantly cluster Forth et al. Surprisingly, PRC1 exhibited no directional preference, generating equivalent frictional forces regardless of microtubule polarity. Might the property of frictional asymmetry be harnessed within active microtubule networks by the cell?
Computer simulations predict, and experiments directly verify, that when a dimeric NuMA construct cross-links microtubules in the parallel geometry and those microtubules are then perturbed by external forces, NuMA can autonomously move toward the microtubule minus ends Forth et al. By taking advantage of this intrinsic mechanical anisotropy, it is possible that within an actively fluctuating microtubule network, directional motion of certain MAPs can be achieved without the requirement that a motor protein directly transport the molecule.
Such fluctuations could result from the competition between plus end— and minus end—directed motor proteins that cross-link the microtubule pair, as described earlier Hentrich and Surrey, Because frictional asymmetry likely arises from the directional dependence of force-induced bond breaking at the atomic level, it may also be the case that this type of autonomous directed motion occurs within other active polymer networks in the cell.
In addition to producing frictional resistance and acting as a brake against motion, a fascinating new mechanism of force generation was recently observed for Ase1, the yeast MAP Experiments suggest that Ase1 cross-linking two microtubules may behave as a compressible gas, generating entropic forces that increase as diffusible molecules of Ase1 are compacted into smaller and smaller overlap regions Fig.
These nonmotor cross-linking proteins undergo thermally driven diffusion along the lattice of each of the microtubule surfaces they contact.
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