Gliding Assays Research Articles

Surface Passivation of Norland Optical Adhesive Improves the Guiding Efficiency of Gliding Microtubules in Microfluidic Devices.

The microtubule-kinesin biomolecular motor system, which is vital for cellular function, holds significant promise for nanotechnological applications. In vitro gliding assays have demonstrated the ability to transport microcargo by propelling microtubules across kinesin-coated surfaces. However, the uncontrolled directional motion of microtubules has posed significant challenges, limiting the system's application for precise cargo delivery. Microfluidic devices provide a means to direct microtubule movement through their geometric features. Norland Optical Adhesive (NOA) is valued for its mold-free application in microfluidic device fabrication; however, microtubules often climb up channel walls, limiting controlled movement. In this study, a surface passivation method for NOA is introduced, using polyethylene glycol via a thiol-ene click reaction. This technique significantly improved the directional control and concentration of microtubules within NOA microchannels. This approach presents new possibilities for the precise application of biomolecular motors in nanotechnology, enabling advancements in the design of microfluidic systems for complex biomolecular manipulations.

KnotResolver: tracking self-intersecting filaments in microscopy using directed graphs.

Quantification of microscopy time series of in vitro reconstituted motor-driven microtubule transport in "gliding assays" is typically performed using computational object tracking tools. However, these are limited to non-intersecting and rod-like filaments. Here, we describe a novel computational image-analysis pipeline, KnotResolver, to track image time series of highly curved self-intersecting looped filaments (knots) by resolving cross-overs. The code integrates filament segmentation and cross-over or "knot" identification based on directed graph representation, where nodes represent cross-overs and edges represent the path connecting them. The graphs are mapped back to contours and the distance to a reference minimized. The accuracy of contour detection is sub-pixel with a robustness to noise. We demonstrate the utility of KnotResolver by automatically quantifying "flagella-like" curvature dynamics and wave-like oscillations of clamped microtubules in a "gliding assay." The MATLAB-based source code is released as OpenSource and is available at https://github.com/CyCelsLab/MTKnotResolver.

Surface Topography Induces and Orients Nematic Swarms of Active Filaments: Considerations for Lab-On-A-Chip Devices.

Surface-bound molecular motors can drive the collective motion of cytoskeletal filaments in the form of nematic bands and polar flocks in reconstituted gliding assays. Although these "swarming transitions" are an emergent property of active filament collisions, they can be controlled and guided by tuning the surface chemistry or topography of the substrate. To date, the impact of surface topography on collective motion in active nematics is only partially understood, with most experimental studies focusing on the escape of a single filament from etched channels. Since the late 1990s, significant progress has been made to utilize the nonequilibrium properties of active filaments and create a range of functional nanodevices relevant to biosensing and parallel computation; however, the complexity of these swarming transitions presents a challenge when attempting to increase filament surface concentrations. In this work, we etch shallow, linear trenches into glass substrates to induce the formation of swarming nematic bands and investigate the mechanisms by which surface topography regulates the two-dimensional (2D) collective motion of driven filamentous actin (F-actin). We demonstrate that nematic swarms only appear at intermediate trench spacings and vanish if the trenches are made too narrow, wide, or tortuous. To rationalize these results, we simulate the F-actin as self-propelled, semiflexible chains subject to a soft, spatially modulated potential that encodes the energetic cost of bending a filament along the edge of a trench. In our model, we hypothesize that an individual filament experiences a penalty when its projected end-to-end distance is smaller than the trench spacing ("bending and turning"). However, chains that span the channel width glide above the trenches in a force- and torque-free manner ("crowd-surfing"). Our simulations demonstrate that collections of filaments form nematic bands only at intermediate trench spacings, consistent with our experimental findings.

Left-right Myosin-Is, Myosin1C, and Myosin1D exhibit distinct single molecule behaviors on the plasma membrane of Drosophila macrophages.

Left-right (LR) asymmetry is crucial for animal development, particularly in Drosophila where LR-asymmetric morphogenesis of organs hinges on cellular-level chirality, termed cell chirality. In this species, two class I myosins, Myosin1D (Myo1D), and Myosin1C (Myo1C), respectively determine dextral (wild type) and sinistral (mirror image) cell chirality. Previous studies demonstrated Myo1D's ability to propel F-actin in leftward circles during in vitro gliding assays, suggesting its mechanochemical role in defining dextral chirality. Conversely, Myo1C propels F-actin without exhibiting LR-directional preference in this assay, suggesting at other properties governing sinistral chirality. Given the interaction of Myo1D and Myo1C with the membrane, we hypothesized that differences in their membrane behaviors might be critical in dictating their dextral or sinistral activities. In this study, employing single-molecule imaging analyses, we investigated the dynamic behaviors of Myo1D and Myo1C on the plasma membrane. Our findings revealed that Myo1C exhibits a significantly greater proportion of slow-diffusing population compared to Myo1D. Importantly, this characteristic was contingent upon both head and tail domains of Myo1C. The distinct diffusion patterns of Myo1D and Myo1C did not exert mutual influence on each other. This divergence in membrane diffusion between Myo1D and Myo1C may be crucial for dictating cell and organ chirality.

Wave-like oscillations of clamped microtubules driven by collective dynein transport

Microtubules (MTs) are observed to move and buckle driven by ATP-dependent molecular motors in both mitotic and interphasic eukaryotic cells as well as in specialized structures such as flagella and cilia with a stereotypical geometry. In previous work, clamped MTs driven by a few kinesin motors were seen to buckle and occasionally flap in what was referred to as flagella-like motion. Theoretical models of active-filament dynamics and a following force have predicted that, with sufficient force and binding-unbinding, such clamped filaments should spontaneously undergo periodic buckling oscillations. However, a systematic experimental test of the theory and reconciliation to a model was lacking. Here, we have engineered a minimal system of MTs clamped at their plus ends and transported by a sheet of dynein motors that demonstrate the emergence of spontaneous traveling-wave oscillations along single filaments. The frequencies of tip oscillations are in the millihertz range and are statistically indistinguishable in the onset and recovery phases. We develop a 2D computational model of clamped MTs binding and unbinding stochastically to motors in a “gliding-assay” geometry. The simulated MTs oscillate with a frequency comparable to experiment. The model predicts the effect of MT length and motor density on qualitative transitions between distinct phases of flapping, regular oscillations, and looping. We develop an effective “order parameter” based on the relative deflection along the filament and orthogonal to it. The transitions predicted in simulations are validated by experimental data. These results demonstrate a role for geometry, MT buckling, and collective molecular motor activity in the emergence of oscillatory dynamics.

Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics

Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in invitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in invitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.

Tuning the collective motion of microtubules by their persistence length.

Collective motion is an emergent phenomenon of active matter. The collective motion of cytoskeleton filaments, such as motor protein-propelled microtubules and actin filaments, results from their continuous collisions and alignments. However, the detailed emergence mechanism of collective behaviors is still unclear. Notably, the persistence length, an intrinsic mechanical property of filaments determining their orientational flexibility, is likely to directly affect the collective motion. Yet the relevant experimental verifications are still significantly lacking. Here, we investigated how the persistence length of microtubules influences their collective motion using high-density motility gliding assays in the presence of methylcellulose. We found that the collective motion of microtubules can be tuned by their persistence lengths. By combining statistical analysis of collision with an agent-based model, we showed that collectively gliding microtubules with different persistence lengths have distinct local interactions. Stiffer microtubules have more durable alignments after collisions than the softer ones. A detailed analysis of the specific microtubule-microtubule interactions found that - in contrast to the earlier studies - it is not a greater likelihood of alignment after collisions but a more durable alignment that gives rise to enhanced bundling. The persistence length-dependent durability of alignment dominates the collective behavior of microtubules. Moreover, it affects phase transition processes and final coordinated patterns formed during the collective motion.

Torque generating properties of Tetrahymena ciliary three-headed outer-arm dynein

Eukaryotic cilia/flagella are cellular bio-machines that drive the movement of microorganisms. Molecular motor axonemal dyneins in the axoneme, which consist of an 9 + 2 arrangement of microtubules, play an essential role in ciliary beating. Some axonemal dyneins have been shown to generate torque coupled with the longitudinal motility of microtubules across an array of dyneins fixed to the coverglass surface, resulting in a corkscrew-like translocation of microtubules. In this study, we performed three-dimensional tracking of a microbead coated with axonemal outer-arm dyneins on a freely suspended microtubule. We found that microbeads coated with multiple outer-arm dyneins exhibited continuous right-handed helical trajectories around the microtubule. This unidirectional helical motion differs from that of other types of cytoplasmic dyneins, which exhibit bidirectional helical motility. We also found that, in an in vitro microtubule gliding assay, gliding microtubules driven by outer-arm dyneins tend to turn to the left, causing a curved path, suggesting that the outer-arm dynein itself is able to rotate on its own axis. Two types of torque generated by the axonemal dyneins, corresponding to the forces used to rotate the microtubule unidirectionally with respect to the long and short axes, may regulate ciliary beating with complex waveforms.

Measurement of the persistence length of cytoskeletal filaments using curvature distributions

Cytoskeletal filaments, such as microtubules and actin filaments, play important roles in the mechanical integrity of cells and the ability of cells to respond to their environment. Measuring the mechanical properties of cytoskeletal structures is crucial for gaining insight into intracellular mechanical stresses and their role in regulating cellular processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring filament deformations, such as Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature distributions can be used as an alternative tool to quantify biofilament deformations, and investigate how the apparent stiffness of filaments depends on the resolution and noise of the imaging system. We present analytical calculations of the scaling curvature distributions as a function of filament discretization, and test our predictions by comparing Monte Carlo simulations with results from existing techniques. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of nonfunctional motors, and calculate the persistence length of these filaments. The presented curvature analysis is significantly more accurate compared with existing approaches for small data sets, and can be readily applied to both in vitro and in vivo filament data through the use of the open-source codes we provide.

Microtubules' local and global order on membrane-bound motor proteins

As part of the cytoskeleton, microtubules (MTs) are vital components of all Eukaryotic cells, important for cell division and intracellular transport. Converting chemical energy into mechanical energy, kinesin motor proteins “walk” on MTs, transporting cargo throughout the cell. In this project, we investigated MT-kinesin-based transportation. We take advantage of the microtubule gliding assay configuration to probe collective behavior in dense MT swarms. Experiments were performed using kinesin motors coupled to either a glass substrate or lipid bilayer.

Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N-Oxide.

The biomolecular motor protein kinesin and its associated filamentous protein microtubule have been finding important nanotechnological applications in the recent years. Rigidity of the microtubules, which are propelled by kinesin motors in an in vitro gliding assay, is an important metric that determines the success of utilization of microtubules and kinesins in various applications, such as transportation, sensing, sorting, molecular robotics, etc. Therefore, regulating the rigidity of kinesin-propelled microtubules has been critical. In this work, we report a simple strategy to regulate the rigidity of kinesin-propelled microtubules in an in vitro gliding assay. We demonstrate that rigidity of the microtubules, propelled by kinesins in an in vitro gliding assay, can be modulated simply by using the natural osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of microtubules can be modulated over a wide range. Based on this strategy, we are able to reduce the persistence length of microtubules, a measure of microtubule rigidity, ∼8 fold by using TMAO at the concentration of 1.5 M. Furthermore, we found that the decreased rigidity of the kinesin-propelled microtubules can be restored upon elimination of TMAO from the in vitro gliding assay. Alteration in the rigidity of microtubules is accounted for by the non-uniformity of the force applied by kinesins along the microtubules in the presence of TMAO. This work offers a facile strategy to reversibly regulate the rigidity of kinesin-propelled microtubules in situ, which would widen the applications of the biomolecular motor kinesin and its associated protein microtubule in various fields.

Dynamic Pattern Formation of Active Matters Triggered by Mechanical Stimuli.

In vitro gliding assay of the filamentous protein microtubule (MT) on a kinesin motor protein coated surface has appeared as a classic platform for studying active matters. At high densities, the gliding MTs spontaneously align and self-organize into fascinating large-scale patterns. Application of mechanical stimuli e.g., stretching stimuli to the MTs gliding on a kinesin-coated surface can modulate their self-organization and patterns according to the boundary conditions. Depending on the mode of stretching, MT at high densities change their moving direction and exhibit various kinds of patterns such as stream, zigzag and vortex pattern. In this chapter, we discuss detail procedures on how to apply mechanical stimuli to the moving MTs on a kinesin coated substrate.

Growth rate-dependent flexural rigidity of microtubules influences pattern formation in collective motion

BackgroundMicrotubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation.ResultsHere, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning.ConclusionOur study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future.Graphic

Prospects for single-molecule electrostatic detection in molecular motor gliding motility assays

Molecular motor gliding motility assays based on myosin/actin or kinesin/microtubules are of interest for nanotechnology applications ranging from cargo-trafficking in lab-on-a-chip devices to novel biocomputation strategies. Prototype systems are typically monitored by expensive and bulky fluorescence microscopy systems. The development of integrated, direct electric detection of single filaments would strongly benefit applications and scale-up. We present estimates for the viability of such a detector by calculating the electrostatic potential change generated at a carbon nanotube transistor by a motile actin filament or microtubule under realistic gliding assay conditions. We combine this with detection limits based on previous state-of-the-art experiments using carbon nanotube transistors to detect catalysis by a bound lysozyme molecule and melting of a bound short-strand DNA molecule. Our results show that detection should be possible for both actin and microtubules using existing low ionic strength buffers given good device design, e.g., by raising the transistor slightly above the guiding channel floor. We perform studies as a function of buffer ionic strength, height of the transistor above the guiding channel floor, presence/absence of the casein surface passivation layer for microtubule assays and the linear charge density of the actin filaments/microtubules. We show that detection of microtubules is a more likely prospect given their smaller height of travel above the surface, higher negative charge density and the casein passivation, and may possibly be achieved with the nanoscale transistor sitting directly on the guiding channel floor.

Two modes of PRC1-mediated mechanical resistance to kinesin-driven microtubule network disruption

The proper organization of the microtubule-based spindle during cell division requires the collective activity of many different proteins. These include non-motor microtubule-associated proteins (MAPs), whose functions include crosslinking microtubules to regulate filament sliding rates and assemble microtubule arrays. One such protein is PRC1, an essential MAP that has been shown to preferentially crosslink overlapping antiparallel microtubules at the spindle midzone. PRC1 has been proposed to act as a molecular brake, but insight into the mechanism of how PRC1 molecules function cooperatively to resist motor-driven microtubule sliding and to allow for the formation of stable midzone overlaps remains unclear. Here, we employ a modified microtubule gliding assay to rupture PRC1-mediated microtubule pairs using surface-bound kinesins. We discovered that PRC1 crosslinks always reduce bundled filament sliding velocities relative to single-microtubule gliding rates and do so via two distinct emergent modes of mechanical resistance to motor-driven sliding. We term these behaviors braking and coasting, where braking events exhibit substantially slowed microtubule sliding compared to coasting events. Strikingly, braking behavior requires the formation of two distinct high-density clusters of PRC1 molecules near microtubule tips. Our results suggest a cooperative mechanism for PRC1 accumulation when under mechanical load that leads to a unique state of enhanced resistance to filament sliding and provides insight into collective protein ensemble behavior in regulating the mechanics of spindle assembly.

Characterization of the motility of monomeric kinesin-5/Cin8

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.

Pattern formation and polarity sorting of driven actin filaments on lipid membranes

Collective motion of active matter is ubiquitously observed, ranging from propelled colloids to flocks of bird, and often features the formation of complex structures composed of agents moving coherently. However, it remains extremely challenging to predict emergent patterns from the binary interaction between agents, especially as only a limited number of interaction regimes have been experimentally observed so far. Here, we introduce an actin gliding assay coupled to a supported lipid bilayer, whose fluidity forces the interaction between self-propelled filaments to be dominated by steric repulsion. This results in filaments stopping upon binary collisions and eventually aligning nematically. Such a binary interaction rule results at high densities in the emergence of dynamic collectively moving structures including clusters, vortices, and streams of filaments. Despite the microscopic interaction having a nematic symmetry, the emergent structures are found to be polar, with filaments collectively moving in the same direction. This is due to polar biases introduced by the stopping upon collision, both on the individual filaments scale as well as on the scale of collective structures. In this context, positive half-charged topological defects turn out to be a most efficient trapping and polarity sorting conformation.

Writhing of Cytokinetic Contractile Rings Reveals that the Contractile Ring is an Elastoporous Cable

We report extraordinary writhing dynamics of isolated fission yeast contractile rings, revealing basic material properties of the actomyosin ring normally hidden under physiological circumstances. Contractile rings exhibited cable-like writhing and coiling, with repeated twisting of the rings leading to multiple coils, similar to the twisting of a telephone cable. Our results suggest the twisting torque is produced by myosin-II in the ring that twisted around anchored actin filaments. Image analysis revealed twisting rates (0.3 rev per micron) very close to those previously reported for myosin II-actin twisting from in vitro gliding assays (0.25 - 2 rev per micron, Nishizaka et al., 1993; Beausang et al., 2008). We show the twisting is quantitatively as would be predicted for a solid cable, and from the measured writhe versus ring length we extracted the effective ratio of ring twisting and bending moduli. A mathematical model quantitatively reproduced the cable-like twisting, based on continuum treatment of the contractile ring as an elastoporous solid, similar to an emerging view of the cell cytoplasm (Moeendarbary et al., 2013). The model predicts that after a transient the ring deforms affinely, with constant twisting rate like a solid cable whose mechanical rigidity is set by its twisted actin filaments. Thus the contractile ring, considered as a whole, is a highly anisotropic elastoporous cable of actin filaments embedded in an effective fluid of myosin-II. We observed these writhing rings in fission yeast cell ghosts, obtained by cell wall digestion and membrane permeabilization that releases cytoplasm (Mishra et al., 2013). Writhing was triggered by sections of contractile rings unanchoring from the weakened membrane, then shortening and writhing due to an apparent twisting torque at the anchored end points. Repeated rotation produced multiple coils in the rings.

Evaluation of Microtubules Binary Collisions using Nano-Patterned Kinesins

Collective motion is a ubiquitous phenomenon manifested in a wide range of living systems ranging from bacterial swarming to animal flocking. The transition from a random distribution to an ordered state resulting in large scale patterns is a fascinating feature demonstrated in collective motion. The reconstituted biomolecular motor system of microtubules propelled by kinesin or dynein motor proteins has been envisioned as an ideal candidate to investigate underlies mechanism of collective motion in vitro. The earlier studies on microtubule/kinesin gliding assay suggest that the alignment of microtubules in binary collisions plays an indispensable factor in achieving collective motion. Therefore, in our ongoing research, we are studying the alignment interactions between microtubule filaments by controlling kinesin number and spacing using gold nanopillars fabricated on the SiO2. To assess the alignment interaction we studied the binary collision events of the microtubules and found colliding microtubules align with each other with high probability at low kinesin spacing. However, with the increase in kinesin spacing microtubules preferred to show overlapping in collisions. Additionally, we measured other parameters related to the binary collisions like the incoming collision angle (i.e., the angle between two approaching microtubules). We found that the distribution of microtubules incoming angle depends on kinesin spacing and arrangements. Our analysis show that at higher spacing microtubules prefer to collide orthogonally and resulted in overlapping events. We also investigated the effect of other parameters like microtubules flexural rigidity and found no significant effects on alignment interactions. Finally, we increased the concentration of microtubules and observed the emergence of bundles and stream patterns in collective motion depending on microtubule concentrations without any influence of depleting or binding agents. This study may provide new insights in understanding the collective motion in nature.

Number Dependence of Microtubule Collective Transport by Kinesin and Dynein

Translational motors that depend on cytoskeletal elements, either actin or tubulin, for their activity are critical for cellular function in eukaryotes. Microtubule (MT)-dependent motors are broadly classified as dyneins and kinesins, based on sequence similarity. Typically, dyneins walk towards the minus-ends of MTs, while kinesins walk towards the plus-ends, with some plant and animal kinesins also seen to be minus-end : The kinetic polarity of MTs with plus indicating net growth and minus net shrinkage. directed. While our understanding of motor mechanics at a single-molecule level has rapidly improved due to developments in force spectroscopy, in vivo motor transport often involves multiple motors acting together. Here, we review our current understanding of collective effects that emerge in motor-driven transport in vivo based on physical mechanisms inferred from in vitro reconstitution experiments involving MT transport, or ‘gliding assays’ : Microtubule (MT) transport by immobilized molecular motors. . We discuss the evidence for number dependence in cargo transport at MT cross-overs, orientation sorting of MTs during axonal regeneration in neurons, spindle bipolarization by MT transport, and nuclear positioning during mitosis in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We discuss how minimal in vitro gliding assays have been successfully used to identify the mechanical properties of collective motor transport that produce such cooperative effects. The ‘loose coupling’ mechanism of Oosawa, which was developed to explain the emergence of cooperation in collective motor transport, appears to be consistent with evidence from kinesin, but not dynein. Additionally, substrate rigidity also appears to play a role in collective force generation, as seen in lipid-anchorage studies of collective transport. Thus, a deeper understanding of the intra- and inter-motor properties of kinesins and dyneins : MT dependent molecular motors that walk predominantly towards the plus- and minus-ends of the MTs respectively. , as well as non-motor effects due to the substrate is required, for the emergence of a complete picture of the in vivo mechanobiology of collective motor transport.