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Marileen Dogterom

Publications and source records attributed to Marileen Dogterom.

15 recordsLinked to original sources

Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach.

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.

Cell Size↗

Assembly dynamics of microtubules at molecular resolution.

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.

Algorithms↗

Continuum description of the cytoskeleton: ring formation in the cell cortex.

Motivated by the formation of ringlike filament structures in the cortex of plant and animal cells, we study the dynamics of a two-dimensional layer of cytoskeletal filaments and motor proteins near a surface by a general continuum theory. As a result of active processes, dynamic patterns of filament orientation and density emerge via instabilities. We show that self-organization phenomena can lead to the formation of stationary and oscillating rings. We present state diagrams that reveal a rich scenario of asymptotic behaviors and discuss the role of boundary conditions.

Biological Clocks↗

Force barriers for membrane tube formation.

We used optical tweezers to measure the force-extension curve for the formation of tubes from giant vesicles. We show that a significant force barrier exists for the formation of tubes, which increases linearly with the radius of the area on which the pulling force is exerted. The tubes form through a first-order transition with accompanying hysteresis. We confirm these results with Monte Carlo simulations and theoretical calculations. Whether membrane tubes can be formed in, for example, biological cells, thus depends on the details of how forces are applied.

Biotin↗

Force generation by dynamic microtubules.

The assembly and disassembly of microtubules can generate pushing and pulling forces that, together with motor proteins, contribute to the correct positioning of chromosomes, mitotic spindles and nuclei in cells. In vitro experiments combined with modeling have shed light on the intrinsic capability of dynamic microtubules to generate force, and various observations of positioning processes in cells and model systems have shown how pushing and pulling forces are used in different situations. A sophisticated set of microtubule-end-binding proteins is responsible for steering dynamic microtubules toward their cellular target and regulating the pushing and/or pulling forces that are generated once contact is established.

Cell Nucleus↗

Three-dimensional control of protein patterning in microfabricated devices.

We present a new procedure that allows for the controlled patterning of functional proteins on the sidewalls of three-dimensional microfabricated chambers. We build microchambers with walls containing gold areas of a defined submicrometer size, to which proteins can be bound specifically, via thiol chemistry. We tested our system by observing the gliding of microtubules along motor-coated microchamber sidewalls and find that introducing an intermediate multilayer of proteins greatly enhances motor protein activity. This combination of spatial confinement and three-dimensional patterning of protein activity opens up new possibilities for the in vitro study of cellular processes, as well as for the development of guided transport mechanisms in nanodevices.

Adsorption↗

Scaling of microtubule force-velocity curves obtained at different tubulin concentrations.

We present a single curve that describes the decay in average growth velocity for microtubules in response to a mechanical force. Curves obtained at two new and one previously studied tubulin concentrations coalesce when normalized with the growth velocity at zero load. This scaling provides direct evidence for a force-independent molecular off rate, in agreement with Brownian ratchet models. In addition, microtubule length changes were measured with a precision up to 10 nm, revealing that microtubules under load abruptly switch between different growth velocities.

Dimerization↗

Microtubules become more dynamic but not shorter during preprophase band formation: a possible "search-and-capture" mechanism for microtubule translocation.

The dynamic behavior of the microtubule cytoskeleton plays a crucial role in cellular organization, but the physical mechanisms underlying microtubule (re)organization in plant cells are poorly understood. We investigated microtubule dynamics in tobacco BY-2 suspension cells during interphase and during the formation of the preprophase band (PPB), the cytoskeletal structure that defines the site of cytokinesis. Here we show that after 2 h of microtubule accumulation in the PPB and concurrent disappearance elsewhere in the cortex, the PPB is completed and starts to breakdown exponentially already 20 min before the onset of prometaphase. During formation of the PPB, the dynamic instability, i.e., the stochastic alternating between growing and shrinking phases, of the cortical microtubules outside the PPB increases significantly, but the microtubules do not become shorter. Based on this, as well as on the cross-linking of microtubules in the PPB and the lack of evidence for motor involvement, we propose a "search-and-capture" mechanism for PPB formation, in which the regulation of dynamic instability causes the cortical microtubules to become more dynamic and possibly longer, while the microtubule cross-linking activity of the developing PPB preferentially stabilizes these "searching" microtubules. Thus, microtubules gradually disappear from the cortex outside the PPB and aggregate to the forming PPB.

Algorithms↗

Active motor proteins can couple cargo to the ends of growing microtubules.

In living cells, dynamic microtubule ends interact with specialized protein complexes located on microtubule targets such as chromosomes and the cell cortex. A significant role in coupling microtubule ends to these complexes has been attributed to motor proteins, which are thought to provide a physical link while at the same time allowing for microtubule growth or shrinkage. In the past, motor-coated beads have been shown to be able to follow the ends of depolymerizing microtubules, in a direction opposite to their natural walking direction. Here we show that beads coated with plus-end-directed motors can also stay attached for several seconds to the ends of growing microtubules. Upon arrival at the microtubule end, fast-moving beads reduce their velocity to the microtubule growth velocity. We show that the tendency to stay attached depends on the initial bead velocity and that the microtubule growth velocity is unaffected by the presence of the bead.

Animals↗

A bending mode analysis for growing microtubules: evidence for a velocity-dependent rigidity.

Microtubules are dynamic protein polymers that continuously switch between elongation and rapid shrinkage. They have an exceptional bending stiffness that contributes significantly to the mechanical properties of eukaryotic cells. Measurements of the persistence length of microtubules have been published since 10 years but the reported values vary over an order of magnitude without an available explanation. To precisely measure the rigidity of microtubules in their native growing state, we adapted a previously developed bending mode analysis of thermally driven shape fluctuations to the case of an elongating filament that is clamped at one end. Microtubule shapes were quantified using automated image processing, allowing for the characterization of up to five bending modes. When taken together with three other less precise measurements, our rigidity data suggest that fast-growing microtubules are less stiff than slow-growing microtubules. This would imply that care should be taken in interpreting rigidity measurements on stabilized microtubules whose growth history is not known. In addition, time analysis of bending modes showed that higher order modes relax more slowly than expected from simple hydrodynamics, possibly by the effects of internal friction within the microtubule.

Algorithms↗

Membrane tube formation from giant vesicles by dynamic association of motor proteins.

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.

Cell Membrane↗

Dynamic instability of microtubules is regulated by force.

Microtubules are long filamentous protein structures that randomly alternate between periods of elongation and shortening in a process termed dynamic instability. The average time a microtubule spends in an elongation phase, known as the catastrophe time, is regulated by the biochemical machinery of the cell throughout the cell cycle. In this light, observed changes in the catastrophe time near cellular boundaries (Brunner, D., and P. Nurse. 2000. Cell. 102:695-704; Komarova, Y.A., I.A. Vorobjev, and G.G. Borisy. 2002. J. Cell Sci. 115:3527-3539) may be attributed to regulatory effects of localized proteins. Here, we argue that the pushing force generated by a microtubule when growing against a cellular object may itself provide a regulatory mechanism of the catastrophe time. We observed an up to 20-fold, force-dependent decrease in the catastrophe time when microtubules grown from purified tubulin were polymerizing against microfabricated barriers. Comparison with catastrophe times for microtubules growing freely at different tubulin concentrations leads us to conclude that force reduces the catastrophe time only by limiting the rate of tubulin addition.

Animals↗

Colloidal epitaxy: playing with the boundary conditions of colloidal crystallization.

We have studied, with quantitative confocal microscopy, epitaxial colloidal crystal growth of particles interacting with an almost hard-sphere (HS) potential in a gravitational field and density matched colloids interacting with a long-range (LR) repulsive potential with a body-centred cubic (BCC) equilibrium crystal phase. We show that in both cases it is possible to grow thick, stacking fault-free metastable crystals: close-packed crystals with any stacking sequence, including hexagonal close packed (HCP), for the HS particles and face-centred cubic (FCC) in the case of the LR colloids. In accordance with recent computer simulations done for HS particles it was found that the optimal lattice constant to grow HS HCP crystals was larger than that of equilibrium FCC crystals. In addition, because of the absence of gravity, pre-freezing could be observed for the particles with the LR potential on a template of charged lines. We also argue that the ability to manipulate colloids with highly focused light, optical traps or tweezers, will become an important tool in both the study of colloidal crystallization and in making new structures. We show how cheap 2D and 3D templates can be made with optical tweezers and demonstrate, in proof of principle experiments with core-shell colloids, how light fields can generate crystal nuclei and other structures in the bulk of concentrated dispersions and how the effect of these structures on the rest of a dispersion can be studied quantitatively in 3D.

Journal Article↗

Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes.

Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubule-organizing center within a cell. Here, we investigate the effect of catastrophes on the dynamics of microtubule asters within microfabricated chambers that mimic the confining geometry of living cells. The use of a glass bead as the microtubule-organizing center allows us to manipulate the aster by using optical tweezers. In the case in which microtubules preexist, we show that because of microtubule buckling, repositioning almost never occurs after relocation with the optical tweezers, although initial microtubule growth always leads the aster to the geometrical center of the chamber. When a catastrophe promoter is added, we find instead that the aster is able to efficiently explore the chamber geometry even after being relocated with the optical tweezers. As predicted by theoretical calculations, the results of our in vitro experiments clearly demonstrate the need for catastrophes for proper positioning in a confining geometry. These findings correlate with recent observations of nuclear positioning in fission yeast cells.

Animals↗

Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth.

In growing Arabidopsis root hairs, the nucleus locates at a fixed distance from the apex, migrates to a random position during growth arrest, and moves from branch to branch in a mutant with branched hairs. Consistently, an artificial increase of the distance between the nucleus and the apex, achieved by entrapment of the nucleus in a laser beam, stops cell growth. Drug studies show that microtubules are not involved in the positioning of the nucleus but that subapical fine F-actin between the nucleus and the hair apex is required to maintain the nuclear position with respect to the growing apex. Injection of an antibody against plant villin, an actin filament-bundling protein, leads to actin filament unbundling and movement of the nucleus closer to the apex. Thus, the bundled actin at the tip side of the nucleus prevents the nucleus from approaching the apex. In addition, we show that the basipetal movement of the nucleus at root hair growth arrest requires protein synthesis and a functional actin cytoskeleton in the root hair tube.

Actins↗