Simple non-fluorescent polarity labeling of microtubules for molecular motor assays (original) (raw)

The role of microtubule movement in bidirectional organelle transport

Proceedings of the National Academy of Sciences, 2008

We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFPtagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks. intracellular transport ͉ molecular motors ͉ kinesin ͉ dynein ͉ cytoskeleton M olecular motor-mediated transport along microtubules is an extensively studied phenomenon in vitro (1-3). Despite significant advances in vitro, understanding how intracellular transport works in vivo still remains one of the big challenges in cell biology. The question of how cellular cargos find their way through the cytoplasm and get targeted to their temporary or final destinations lies at the heart of the problem. One of the major puzzles in this context is the so-called bidirectional organelle transport. The majority of cargos in the cell move in a bidirectional and often remarkably symmetric manner (4, 5). Despite the known kinetic and dynamic asymmetry of the underlying plus-and minus-end directed microtubule motors, the vesicles seem to move with the same rates and run length distributions, and exhibit identical stalling forces, in each direction (4-7). Furthermore, inhibition of transport in one direction typically results in the inhibition of movement in the opposite direction as well (4-11).

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

Physics Reports, 2015

Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized.

Mechanical Properties of Organelles Driven by Microtubule-Dependent Molecular Motors in Living Cells

PLoS ONE, 2011

The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is , one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules. Citation: Bruno L, Salierno M, Wetzler DE, Despó sito MA, Levi V (2011) Mechanical Properties of Organelles Driven by Microtubule-Dependent Molecular Motors in Living Cells. PLoS ONE 6(4): e18332.

Evidence for a Novel Affinity Mechanism of Motor-assisted Transport Along Microtubules

Molecular Biology of the Cell, 2000

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (Ϫ) or (ϩ) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that ␤ tubulin is exposed at the (ϩ) end of the MT.

Cargo transport: molecular motors navigate a complex cytoskeleton

Current Opinion in Cell Biology, 2008

Intracellular cargo transport requires microtubule-based motors, kinesin and cytoplasmic dynein, and the actin-based myosin motors to maneuver through the challenges presented by the filamentous meshwork that comprises the cytoskeleton. Recent in vitro single molecule biophysical studies have begun to explore this process by characterizing what occurs as these tiny molecular motors happen upon an intersection between two cytoskeletal filaments. These studies, in combination with in vivo work, define the mechanism by which molecular motors exchange cargo while traveling between filamentous tracks and deliver it to its destination when going from the cell center to the periphery and back again.

Analysis of persistence during intracellular actin-based transport mediated by molecular motors

Journal of Physics: Conference Series, 2010

The displacement of particles or probes in the cell cytoplasm as a function of time is characterized by different anomalous diffusion regimes. The transport of large cargoes, such as organelles, vesicles or large proteins, involves the action of ATP-consuming molecular motors. We investigate the motion of pigment organelles driven by myosin-V motors in Xenopus laevis melanocytes using a high spatio-temporal resolution tracking technique. By analyzing the turning angles (φ) of the obtained 2D trajectories as a function of the time lag, we determine the critical time of the transition between anticorrelated and directed motion as the time when the turning angles begin to concentrate around φ = 0. We relate this transition with the crossover from subdiffusive to superdiffusive behavior observed in a previous work . We also assayed the properties of the trajectories in cells with inhibited myosin activity, and we can compare the results in the presence and absence of active motors.

Vesicle Movements and Microtubule-Based Motors

Journal of Cell Science, 1986

The movements of many cytoplasmic vesicles follow the paths of microtubules, some moving in one direction and others moving in the opposite direction on the same microtubule. Recently we have isolated one cytoplasmic motor, kinesin, and defined another, the axoplasmic retrograde factor, both of which are capable of powering anionic latex beads in both directions along polar microtubule arrays. Evidence summarized here supports but does not prove the hypothesis that kinesin and the retrograde motors are indeed responsible for powering vesicle movements.

Hither and yon: a review of bi-directional microtubule-based transport

2004

Active transport is critical for cellular organization and function, and impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is increasingly clear that many cargos are moved by both sets of motors, and frequently reverse course. This review compares this bi-directional transport to the more well studied uni-directional transport. It discusses some bi-directionally moving cargos, and critically evaluates three different physical models for how such transport might occur. It then considers the evidence for the number of active motors per cargo, and how the net or average direction of transport might be controlled. The likelihood of a complex linking the activities of kinesin and dynein is also discussed. The paper concludes by reviewing elements of apparent universality between different bi-directionally moving cargos and by briefly considering possible reasons for the existence of bi-directional transport.

The reciprocal coordination and mechanics of molecular motors in living cells

2009

Molecular motors in living cells are involved in whole-cell locomotion, contractility, developmental shape changes, and organelle movement and positioning. Whether motors of different directionality are functionally coordinated in cells or operate in a semirandom ''tug of war'' is unclear. We show here that anterograde and retrograde microtubule-based motors in the flagella of Chlamydomonas are regulated such that only motors of a common directionality are engaged at any single time. A laser trap was used to position microspheres on the plasma membrane of immobilized paralyzed Chlamydomonas flagella. The anterograde and retrograde movements of the microsphere were measured with nanometer resolution as microtubule-based motors engaged the transmembrane protein FMG-1. An average of 10 motors acted to move the microsphere in either direction. Reversal of direction during a transport event was uncommon, and quiescent periods separated every transport event, suggesting the coordinated and exclusive action of only a single motor type. After a jump to 32°C, temperature-sensitive mutants of kinesin-2 (fla10) showed exclusively retrograde transport events, driven by 7 motors on average. These data suggest that molecular motors in living cells can be reciprocally coordinated to engage simultaneously in large numbers and for exclusive transport in a single direction, even when a mixed population of motors is present. This offers a unique model for studying the mechanics, regulation, and directional coordination of molecular motors in a living intracellular environment. Chlamydomonas ͉ dynein ͉ flagella ͉ kinesin-2 ͉ laser trap