Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules (original) (raw)
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Mechanics of Microtubule-Based Membrane Extension
Physical Review Letters, 1997
We observe quasistatic deformation of lipid vesicles from within, due to the polymerization of confined microtubules. A pair of long, narrow membrane sleeves appears, sheathing the microtubule ends as they grow. Spontaneous buckling reveals that the force generated can be greater than 2 pN. The evolution of shape and magnitude of force are consistent with a simple theory for the membrane free energy. We consider a model of the force generating mechanism in which thermal fluctuations of the membrane are "rectified" by the binding of tubulin dimers to the microtubule end.
Protoplasma, 1990
Video-enhanced microscopy was used to study the behavior of cytoplasmic microtubules in flattened reticulopodia of the marine protist Allogromia. Linear microtubule bundles were observed bending to various degrees and then straightening. When microtubules bent sufficiently to contact the plasma membrane, protuberances extended from the pseudopodial margins. These protuberances withdrew as the bent microtubules straightened. In extreme cases, microtubules formed c-shaped loops which moved laterally through the cytoplasm and contacted fenestrae formed within the flattened pseudopodia. A given fenestra first deformed at the site of microtubule contact and then closed as the loop continued its motion; reversal of the microtubule motion reopened the fenestra. By electron microscopy, microtubules are consistently seen within 20 nm of the plasma membrane and are often connected to the membrane by detergent-resistant crosslinks. Together, these observations indicate that microtubule movements can deform the plasma membrane and thus mediate certain aspects of cellular morphogenesis.
The effect of motor-induced shaft dynamics on microtubule stability and length
Biophysical Journal
Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depends on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in presence of kinesin-1 molecular motors. Although the average microtubule dynamics can be captured by an effective rescue rate, the dynamics of individual microtubules differs dramatically when rescue occurs only at exchange sites. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network. SIGNIFICANCE The microtubule network is essential for vital cellular processes like the organization of intracellular transport and division. Although microtubule assembly occurs at its tips, it has recently been reported that tubulin is exchanged along the microtubule shaft. Tubulin exchange plays an essential role in regulating microtubule dynamics and can be induced by molecular motors. Here, we provide the first systematic study of the impact of shaft dynamics on the regulation of rescue events, where a microtubule switches from shrinking to growing. Our results illustrate how the usage of microtubules as tracks for intracellular transport regulates the microtubule network and thus offers a novel perspective on intracellular organization.
Nature Communications, 2019
Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property ...
Membrane tube formation from giant vesicles by dynamic association of motor proteins
Proceedings of the National Academy of Sciences, 2003
The tubular morphology of intracellular membranous compartments is actively maintained through interactions with motor proteins and the cytoskeleton. Moving along cytoskeletal elements, motor proteins exert forces on the membranes to which they are attached, resulting in the formation of membrane tubes and tubular networks. To study the formation of membrane tubes by motor proteins, we developed an in vitro assay consisting of purified kinesin proteins directly linked to the lipids of giant unilamellar vesicles. When the vesicles are brought into contact with a network of immobilized microtubules, membrane tubes and tubular networks are formed. Through systematic variation of the kinesin concentration and membrane composition we study the mechanism involved. We show that a threshold concentration of motor proteins is needed and that a low membrane tension facilitates tube formation. Forces involved in tube formation were measured directly with optical tweezers and are shown to depend only on the tension and bending rigidity of the membrane. The forces were found to be higher than can be generated by individual motor proteins, indicating that multiple motors were working together to pull tubes. We propose a simple mechanism by which individual motor proteins can dynamically associate into clusters that provide the force needed for the formation of tubes, explaining why, in contrast to earlier findings [Roux, A., Cappello, G., Cartaud, J., Prost, J., Goud, B. & Bassereau, P. (2002) Proc. Natl. Acad. Sci. USA 99, 5394 -5399], motor proteins do not need to be physically linked to each other to be able to pull tubes.
Preparation of segmented microtubules to study motions driven by the disassembling microtubule ends
Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.
Dynamics of microtubules: highlights of recent computational and experimental investigations
Journal of physics. Condensed matter : an Institute of Physics journal, 2017
Microtubules are found in most eukaryotic cells, with homologs in eubacteria and archea, and they have functional roles in mitosis, cell motility, intracellular transport, and the maintenance of cell shape. Numerous efforts have been expended over the last two decades to characterize the interactions between microtubules and the wide variety of microtubule associated proteins that control their dynamic behavior in cells resulting in microtubules being assembled and disassembled where and when they are required by the cell. We present the main findings regarding microtubule polymerization and depolymerization and review recent work about the molecular motors that modulate microtubule dynamics by inducing either microtubule depolymerization or severing. We also discuss the main experimental and computational approaches used to quantify the thermodynamics and mechanics of microtubule filaments.
Length control of microtubules by depolymerizing motor proteins
Europhysics Letters (epl), 2008
In many intracellular processes, the length distribution of microtubules is controlled by depolymerizing motor proteins. Experiments have shown that, following non-specific binding to the surface of a microtubule, depolymerizers are transported to the microtubule tip(s) by diffusion or directed walk and, then, depolymerize the microtubule from the tip(s) after accumulating there. We develop a quantitative model to study the depolymerizing action of such a generic motor protein, and its possible effects on the length distribution of microtubules. We show that, when the motor protein concentration in solution exceeds a critical value, a steady state is reached where the length distribution is, in general, non-monotonic with a single peak. However, for highly processive motors and large motor densities, this distribution effectively becomes an exponential decay. Our findings suggest that such motor proteins may be selectively used by the cell to ensure precise control of MT lengths. The model is also used to analyze experimental observations of motor-induced depolymerization.
Membrane-mediated interactions induce spontaneous filament bundling
2018
The plasma membrane and cytoskeleton of living cells are closely coupled dynamical systems. Internal cytoskeletal elements such as actin filaments and microtubules continually exert forces on the membrane, resulting in the formation of membrane protrusions. In this paper we investigate the interplay between the shape of a cell distorted by pushing and pulling forces generated by microtubules and the resulting rearrangement of the microtubule network. From analytical calculations, we find that two microtubules that deform the vesicle can both attract or repel each other, depending on their angular separations and the direction of the imposed forces. We also show how the existence of attractive interactions between multiple microtubules can be deduced analytically, and further explore general interactions through Monte Carlo simulations. Our results suggest that the commonly reported parallel structures of microtubules in both biological and artificial systems can be a natural consequ...