Leslie Conway

MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis

Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein–dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasis.

Motor transport of self-assembled cargos in crowded environments

Intracellular transport of cargo particles is performed by multiple motors working in concert. However, the mechanism of motor association to cargos is unknown. It is also unknown how long individual motors stay attached, how many are active, and how multimotor cargos would navigate a densely crowded filament with many other motors. Prior theoretical and experimental biophysical model systems of intracellular cargo have assumed fixed teams of motors transporting along bare microtubules or microtubules with fixed obstacles. Here, we investigate a regime of cargos transporting along microtubules crowded with free motors. Furthermore, we use cargos that are able to associate or dissociate motors as it translocates. We perform in vitro motility reconstitution experiments with high-resolution particle tracking. Our model system consists of a quantum dot cargo attached to kinesin motors, and additional free kinesin motors that act as traffic along the microtubule. Although high densities of kinesin motors hinder forward motion, resulting in a lower velocity, the ability to associate motors appears to enhance the run length and attachment time of the quantum dot, improving overall cargo transport. These results suggest that cargos that can associate new motors as they transport could overcome traffic jams.

Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65

Microtubules are rigid, proteinaceous filaments required to organize and rearrange the interior of cells. They organize space by two mechanisms, including acting as the tracks for long-distance cargo transporters, such as kinesin-1, and by forming a network that supports the shape of the cell. The microtubule network is composed of microtubules and a bevy of associated proteins and enzymes that self-organize using non-equilibrium dynamic processes. In order to address the effects of self-organization of microtubules, we have utilized the filament-gliding assay with kinesin-1 motors driving microtubule motion. To further enhance the complexity of the system and determine if new patterns are formed, we added the microtubule crosslinking protein MAP65-1. MAP65-1 is a microtubule-associated protein from plants that crosslinks antiparallel microtubules, similar to mammalian PRC1 and fission yeast Ase1. We find that MAP65 can slow and halt the velocity of microtubules in gliding assays, but when pre-formed microtubule bundles are added to gliding assays, kinesin-1 motors can pull apart the bundles and reconstitute cell-like protrusions.

Microtubule orientation and spacing within bundles is critical for long-range kinesin-1 motility.

Cells rely on active transport to quickly organize cellular cargo. How cells regulate transport is not fully understood. One proposed mechanism is that motor activity could be altered through the architecture of the cytoskeleton. This mechanism is supported by the fact that the cytoskeletal network is tightly regulated in cells and filament polarity within networks dictates motor directionality. For instance, axons contain bundles of parallel microtubules and all cargos with the same motor species will move in the same direction. It is not clear how other types of networks, such as antiparallel bundles in dendrites, can regulate motor transport. To understand how the organization of microtubules within bundles can regulate transport, we studied kinesin‐1 motility on three bundle types: random‐polarity bundles that are close‐packed, parallel polarity bundles, and antiparallel polarity bundles that are spaced apart. We find that close‐packed bundles inhibit motor motion, while parallel arrays support unidirectional motion. Spacing the microtubules with microtubule‐associated proteins enhances run lengths. Our results indicate that microtubule bundle architecture dictates the motion of single motors and could have effects on cargo transport.

Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.

A model system to study transport of self-assembled cargos

Intracellular transport is the process by which cellular cargos, such as organelles and proteins, are moved throughout the cell. Motor proteins bind these cargos and walk along microtubule tracks to deliver them to specific regions of the cell. In axons, cargos are transported by either fast or slow axonal transport. Fast axonal transport is performed by fixed teams of motors bound to membranous cargos, whereas slow axonal transport is thought to be performed by motors that transiently self-assemble with cargos, assembling and disassembling throughout transport. While recent studies have begun to shed light on the nature of slow axonal transport, there are many open questions about the mechanism of action for transient motor association, and how they could result in effective, yet slow, long-range transport. Here, we describe an in vitro system to study self-assembled cargos using quantum dots (Qdots) as artificial cargos. In this system, kinesin motors are able to form transient interactions with Qdot cargos, allowing for the study of self-assembled cargos that assemble and disassemble during transport. Using this system, we can begin to probe the effects of self-assembly on cargo transport properties.

Measuring Transport of Motor Cargos

In this chapter, we describe experimental techniques used in vitro to illuminate how small teams of motors can work to translocate cargos. We will focus on experiments utilizing in vitro reconstitution, artificial or ex vivo purified cargos, and fluorescence imaging. A number of studies have been able to recapitulate the activities of cargo transport driven by small teams of motors elucidating how multiple motors can work together to transport cargos within the cell. Here, we describe some of the methods employed and highlight important experimental details needed to perform these experiments.

Characterization of Kinesin-1 E157A and E157K Mutants

Kinesin-1 is a microtubule-based cargo-transporting motor that plays an important role in axonal transport. Kinesin is responsible for bringing newly synthesized materials from the cell body to the axon terminal in order to properly maintain long axonal processes. Numerous mutations in the kinesin-1 gene have been found to affect the ability of these motors to transport vital material to the axon terminal, resulting in neuronal degeneration. Kinesin possesses two motor domains, each of which contains an ATP binding pocket as well as a microtubule-binding domain. Here we study two mutations in the human kinesin gene, E157A and E157K, shown to inhibit development in flies. This residue position is located near the microtubule-binding site on the motor domain. We find that, in a filament gliding assay, microtubule gliding velocities are reduced for kinesin E157A and E157K mutants compared to wild type kinesin. Interestingly, single molecule studies using these same mutants do not show a reduced velocity compared to wild type. Single molecule studies also reveal that while these mutants have velocities identical to wild type kinesin, they show reduced run lengths. The difference in these two assays is that kinesin motors must coordinate in the filament gliding assay, but not in the single-molecule assays. All together, these results suggest that in addition to a defect in processivity, these mutants possess a cooperativity defect, resulting in slow motility only when groups of mutant motors must work together. These results are significant because, in the cell, multiple motors transport cargos, so defects in coordination between motors are a unique mechanism to inhibit transport.

Biophysical Analysis of Microtubule Motor Traffic Jams

The microtubule motor proteins, kinesin-1 and cytoplasmic dynein, are essential to transport cargo along microtubule tracks throughout the cell. This process relies on the processive nature of motor proteins. Single molecule studies show that both kinesin-1 and cytoplasmic dynein travel an average of 1 µm in distance with a velocity of ∼0.6 µm/s in vitro. While these studies accurately depict the properties of these motors, they do not take into account the crowded environment these motors face in vivo. In contrast to dilute single molecule experiments, motors likely encounter numerous obstacles along the microtubule in cells. These obstacles could include other motors or microtubule associated proteins that bind along the microtubule, blocking the path needed by kinesin-1 or cytoplasmic dynein to continue their processive run. The objective of this study is to investigate how obstacles affect motor properties. We use increasing concentrations of the same or opposite motor to form obstacles in vitro. Motors are tagged with quantum dots to eliminate the issue of photobleaching and allow visualization of entire run lengths via total internal reflection fluorescence (TIRF) microscopy. Experiments are carried out using labeled motors in the presence of increasing concentrations of unlabeled motors. Processivity, velocity, dwell time, and pauses were measured for kinesin-1 across a concentration range of 1 nM-200 nM. We see that while velocity decreases in crowded conditions, the dwell time increases. Run length is seen to increase between 5 and 10 nM. In addition, pause frequency decreases in crowded conditions, while the average pause duration increases. We conclude that kinesin-1 motor properties are regulated by levels of crowding along the microtubule.