Kinesin Recycling in Stationary Membrane Tubes

Collections of motors dynamically organize to extract membrane tubes. These tubes grow but often pause or change direction as they traverse an underlying microtubule (MT) network. In vitro, membrane tubes also stall: they stop growing in length despite a large group of motors available at the tip to pull them forward. In these stationary membrane tubes in vitro, we find that clusters of processive kinesin motors form and reach the tip of the tube at regular time intervals. The average times between cluster arrivals depends on the time over which motors depart from the tip, suggesting that motors are recycled toward the tip. Numerical simulations of the motor dynamics in the membrane tube and on the MTs show that the presence of cooperative binding between motors quantitatively accounts for the clustering observed experimentally. Cooperative binding along the length of the MT and a nucleation point at a distance behind the tip define the recycling period. Based on comparison of the numerical results and experimental data, we estimate a cooperative binding probability and concentration regime where the recycling phenomenon occurs.     

Mechanism of membrane nanotube formation by molecular motors

Membrane nanotubes are ubiquitous in eukaryotic cells due to their involvement in the communication between many different membrane compartments. They are very dynamical structures, which are generally extended along the microtubule network. One possible mechanism of tube formation involves the action of molecular motors, which can generate the necessary force to pull the tubes along the cytoskeleton tracks. However, it has not been possible so far to image in living organisms simultaneously both tube formation and the molecular motors involved in the process. The reasons for this are mainly technological. To overcome these limitations and to elucidate in detail the mechanism of tube formation, many experiments have been developed over the last years in cell-free environments. In the present review, we present the results, which have been obtained in vitro either in cell extracts or with purified and artificial components. In particular, we will focus on a biomimetic system, which involves Giant Unilamellar Vesicles, kinesin-1 motors and microtubules in the presence of ATP. We present both theoretical and experimental results based on fluorescence microscopy that elucidate the dynamics of membrane tube formation, growth and stalling.     

Nonprocessive Motor Dynamics at the Microtubule Membrane Tube Interface

Key cellular processes such as cell division, membrane compartmentalization, and intracellular transport rely on motor proteins. Motors have been studied in detail on the single motor level such that information on their step size, stall force, average run length, and processivity are well known. However, in vivo, motors often work together, so that the question of their collective coordination has raised great interest. Here, we specifically attach motors to giant vesicles and examine collective motor dynamics during membrane tube formation. Image correlation spectroscopy reveals directed motion as processive motors walk at typical speeds (≤500 nm/s) along an underlying microtubule and accumulate at the tip of the growing membrane tube. In contrast, nonprocessive motors exhibit purely diffusive behavior, decorating the entire length of a microtubule lattice with diffusion constants at least 1000 times smaller than a freely-diffusing lipid-motor complex in a lipid bilayer (1 μm²/s); fluorescence recovery after photobleaching experiments confirm the presence of the slower-moving motor population at the microtubule-membrane tube interface. We suggest that nonprocessive motors dynamically bind and unbind to maintain a continuous interaction with the microtubule. This dynamic and continuous interaction is likely necessary for nonprocessive motors to mediate bidirectional membrane tube dynamics reported previously.     

Microtubule motor proteins and the organization of the pollen tube cytoplasm

Molecular motors are molecules that drive a wide range of activities (for example, organelle movement, chromosome segregation, and flagellar movement) in cells. Thus, they play essential roles in diverse cellular functions. Understanding their structures, mechanisms of action and different roles is therefore of great practical importance. The role of microtubules during pollen tube growth is presently not identified, nor are basic properties. We do not know, for example, where microtubules are organized, the extent of microtubule dynamics, and the polarity of microtubules in the pollen tube. Roles of microtubules and related motors in organelle trafficking are not clear. Regardless of scarce information, microtubule-based motors of both the kinesin and dynein families have been identified in the pollen tube. Most of these microtubule motors have also been found in association with membrane-bounded organelles, which suggest that these proteins could translocate organelles or vesicles along microtubules. The biochemical features of these proteins are typical of the motor protein class. Immunofluorescence microscopy of pollen tubes probed with antibodies that cross-react with microtubule motors indicate that these proteins are localized in different regions of the pollen tube; therefore, they could have different roles. Although a number of microtubule motors have been identified in the pollen tube, the role of these proteins during pollen tube germination and growth or organelle movement is not yet recognized, as tube elongation and organelle movement in the pollen tube depend mostly on actin filaments. In the effort to understand the specific role that microtubules and related motors have in the pollen tube, it is therefore necessary to identify the molecular machinery that interacts with microtubules. Furthermore, it is crucial to clearly establish the types of interaction between organelles and microtubules. This review summarizes the current state of the art on microtubule motors in the pollen tube, mainly surrounding the putative roles of microtubule motors in organelle movement and cytoplasmic organization. Some hypotheses and speculations are also presented.     

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.     

Torque generation of kinesin motors is governed by the stability of the neck domain

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules. In stark contrast to kinesin-1, these motors display a wide range of left-handed spiraling around microtubules and thus generate torque during cargo transport. We provide direct evidence that kinesin's neck region determines the torque-generating properties. A model system based on kinesin-1 corroborates this result: disrupting the stability of the neck by inserting flexible peptide stretches resulted in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduced the spiraling of the motor up to the point of protofilament tracking. Finally, we present a model that explains the physical basis of kinesin's spiraling around the microtubule.     

The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.     

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

Molecular motors translocate along cytoskeletal filaments, as in the case of kinesin motors on microtubules. Although conventional kinesin-1 tracks a single microtubule protofilament, other kinesins, akin to dyneins, switch protofilaments. However, the molecular trajectory—whether protofilament switching occurs in a directed or stochastic manner—is unclear. Here, we used high-resolution optical tweezers to track the path of single budding yeast kinesin-8, Kip3, motor proteins. Under applied sideward loads, we found that individual motors stepped sideward in both directions, with and against loads, with a broad distribution in measured step sizes. Interestingly, the force response depended on the direction. Based on a statistical analysis and simulations accounting for the geometry, we propose a diffusive sideward stepping motion of Kip3 on the microtubule lattice, asymmetrically biased by force. This finding is consistent with previous multimotor gliding assays and sheds light on the molecular switching mechanism. For kinesin-8, the diffusive switching mechanism may enable the motor to bypass obstacles and reach the microtubule end for length regulation. For other motors, such a mechanism may have implications for torque generation around the filament axis.     

Directional loading of the kinesin motor molecule as it buckles a microtubule.

Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.     

Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional single-bead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport.     

Microtubule motors and pollen tube growth—still an open question

The growth of pollen tubes is supported by the continuous supply of secretory vesicles in the tip area. Movement and accumulation of vesicles is driven by the dynamic interplay between the actin cytoskeleton and motor proteins of the myosin family. A combination of the two protein systems is also responsible for the bidirectional movement of larger organelle classes. In contrast, the role of microtubules and microtubule-based motors is less clear and often ambiguous. Nevertheless, there is evidence which shows that the pollen tube contains a number of microtubule-based motors of the kinesin family. These motor proteins are likely to be associated with pollen tube organelles and, consequently, they have been hypothesized to participate in the distribution of organelles during pollen tube growth. Whether microtubule motor proteins take part in either the transport or positioning of organelles is not known for sure, but there is evidence for this second possibility. This review will discuss the current knowledge of microtubule-based motor proteins (including kinesins and hypothetical dyneins) and will make some hypothesis about their role in the pollen tube.     

Regularity and Synchrony in Motor Proteins

We investigate the origin of the regularity and synchrony which have been observed in numerical experiments of two realistic models of molecular motors, namely Fisher–Kolomeisky’s model of myosin V for vesicle transport in cells and Duke’s model of myosin II for sarcomere shortening in muscle contraction. We show that there is a generic organizing principle behind the emergence of regular gait for a motor pulling a large cargo and synchrony of action of many motors pulling a single cargo. These results are surprising in that the models used are inherently stochastic, and yet they display regular behaviors in the parameter range relevant to experiments. Our results also show that these behaviors are not tied to the particular models used in these experiments, but rather are generic to a wide class of motor protein models.     

Membrane/microtubule tip attachment complexes (TACs) allow the assembly dynamics of plus ends to push and pull membranes into tubulovesicular networks in interphase Xenopus egg extracts

We discovered by using high resolution video microscopy, that membranes become attached selectively to the growing plus ends of microtubules by membrane/microtubule tip attachment complexes (TACs) in interphase- arrested, undiluted, Xenopus egg extracts. Persistent plus end growth of stationary microtubules pushed the membranes into thin tubules and dragged them through the cytoplasm at the approximately 20 microns/min velocity typical of free plus ends. Membrane tubules also remained attached to plus ends when they switched to the shortening phase of dynamic instability at velocities typical of free ends, 50-60 microns/min. Over time, the membrane tubules contacted and fused with one another along their lengths, forming a polygonal network much like the distribution of ER in cells. Several components of the membrane networks formed by TACs were identified as ER by immunofluorescent staining using antibodies to ER-resident proteins. TAC motility was not inhibited by known inhibitors of microtubule motor activity, including 5 mM AMP-PNP, 250 microM orthovanadate, and ATP depletion. These results show that membrane/microtubule TACs enable polymerizing ends to push and depolymerizing ends to pull membranes into thin tubular extensions and networks at fast velocities.     

Role of curvature and phase transition in lipid sorting and fission of membrane tubules

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.     

Effects of dynein on microtubule mechanics and centrosome positioning

To determine forces on intracellular microtubules, we measured shape changes of individual microtubules following laser severing in bovine capillary endothelial cells. Surprisingly, regions near newly created minus ends increased in curvature following severing, whereas regions near new microtubule plus ends depolymerized without any observable change in shape. With dynein inhibited, regions near severed minus ends straightened rapidly following severing. These observations suggest that dynein exerts a pulling force on the microtubule that buckles the newly created minus end. Moreover, the lack of any observable straightening suggests that dynein prevents lateral motion of microtubules. To explain these results, we developed a model for intracellular microtubule mechanics that predicts the enhanced buckling at the minus end of a severed microtubule. Our results show that microtubule shapes reflect a dynamic force balance in which dynein motor and friction forces dominate elastic forces arising from bending moments. A centrosomal array of microtubules subjected to dynein pulling forces and resisted by dynein friction is predicted to center on the experimentally observed time scale, with or without the pushing forces derived from microtubule buckling at the cell periphery.     

The axonal transport motor kinesin‐2 navigates microtubule obstacles via protofilament switching

Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule‐associated proteins (MAPs). Much attention has focused on the behavior of kinesin‐1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin‐2 in axonal transport. We have previously shown that, unlike kinesin‐1, kinesin‐2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin‐2 efficiently navigates Tau on the microtubule surface is unknown. We hypothesized that mammalian kinesin‐2 side‐steps to adjacent protofilaments to maneuver around MAPs. To test this, we used single‐molecule imaging to track the characteristic run length and protofilament switching behavior of kinesin‐1 and kinesin‐2 motors in the absence and presence of 2 different microtubule obstacles. Under all conditions tested, kinesin‐2 switched protofilaments more frequently than kinesin‐1. Using computational modeling that recapitulates run length and switching frequencies in the presence of varying roadblock densities, we conclude that kinesin‐2 switches protofilaments to navigate around microtubule obstacles. Elucidating the kinesin‐2 mechanism of navigation on the crowded microtubule surface provides a refined view of its contribution in facilitating axonal transport.      

On and Around Microtubules: An Overview

Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.     

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

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.     

How molecular motors are arranged on a cargo is important for vesicular transport

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself--and motor organization on the cargo--affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their 'on' rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.