Self-organization Of Microtubules Research Articles

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.

The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

Towards high throughput production of artificial egg oocytes using microfluidics

The production of micron-size droplets using microfluidic tools offers new opportunities to carry out biological assays in a controlled environment. We apply these strategies by using a flow-focusing microfluidic device to encapsulate Xenopus egg extracts, a biological system recapitulating key events of eukaryotic cell functions in vitro. We present a method to generate monodisperse egg extract-in-oil droplets and use high-speed imaging to characterize the droplet pinch-off dynamics leading to the production of trains of droplets. We use fluorescence microscopy to show that our method does not affect the biological activity of the encapsulated egg extract by observing the self-organization of microtubules and actin filaments, two main biopolymers of the cell cytoskeleton, encapsulated in the produced droplets. We anticipate that this assay might be useful for quantitative studies of biological systems in a confined environment as well as high throughput screenings for drug discovery.

Exposure of biological preparations to radiofrequency electromagnetic fields under low gravity

There is interest as to whether the electromagnetic fields used in mobile radiotelephony might affect biological processes. Other weak fields such as gravity intervene in a number of physical and biological processes. Under appropriate in vitro conditions, the macroscopic self-organization of microtubules, a major cellular component, is triggered by gravity. We wished to investigate whether self-organization might also be affected by radiotelephone electromagnetic fields. Detecting a possible effect requires removing the obscuring effects triggered by gravity. A simple manner of doing this is by rotating the sample about the horizontal. However, if the external field does not also rotate with the sample, its possible effect might also be averaged down by rotation. Here, we describe an apparatus in which both the sample and an applied radiofrequency electromagnetic field (1.8 GHz) are stationary with respect to one another while undergoing horizontal rotation. The electromagnetic field profile within the apparatus has been measured and the apparatus tested by reproducing the in vitro behavior of microtubule preparations under conditions of weightlessness. Specific adsorption rates of electromagnetic energy within a sample are measured from the initial temperature rise the incident field causes. The apparatus can be readily adapted to expose samples to various other external fields and factors under conditions of weightlessness.

Wood Cell-Wall Structure Requires Local 2D-Microtubule Disassembly by a Novel Plasma Membrane-Anchored Protein

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern.

Motor-Mediated Microtubule Self-Organization in Dilute and Semi-Dilute Filament Solutions

We study molecular motor-induced microtubule self-organization in dilute and semi-dilute filament solutions. In the dilute case, we use a probabilistic model of microtubule interaction via molecular motors to investigate microtubule bundle dynamics. Microtubules are modeled as polar rods interacting through fully inelastic, binary collisions. Our model indicates that initially disordered systems of interacting rods exhibit an orientational instability resulting in spontaneous ordering. We study the existence and dynamic interaction of microtubule bundles analytically and numerically. Our results reveal a long term attraction and coalescing of bundles indicating a clear coarsening in the system; microtubule bundles concentrate into fewer orientations on a slow logarithmic time scale. In semi-dilute filament solutions, multiple motors can bind a filament to several others and, for a critical motor density, induce a transition to an ordered phase with a nonzero mean orientation. Motors attach to a pair of filaments and walk along the pair bringing them into closer alignment. We develop a spatially homogenous, mean-field theory that explicitly accounts for a force-dependent detachment rate of motors, which in turn affects the mean and the fluctuations of the net force acting on a filament. We show that the transition to the oriented state can be both continuousmore » and discontinuous when the force-dependent detachment of motors is important.« less

A Three-Dimensional Computer Simulation Model Reveals the Mechanisms for Self-Organization of Plant Cortical Microtubules into Oblique Arrays

The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT interactions, boundary conditions, and the bundling cutoff angle impact the rate and extent of CMT organization, whereas branch-form CMT nucleation did not significantly impact the rate of CMT organization but was necessary to generate polarity during CMT organization. We also found that the dynamic instability parameters from twisted growth mutants were not sufficient to generate oblique CMT arrays. Instead, we found that parameters regulating branch-form CMT nucleation and boundary conditions at the end walls are important for forming oblique CMT arrays. Together, our computer model provides new mechanistic insights into how plant CMTs self-organize into specific 3D arrangements.

Microtubule Motility on Reconstituted Meiotic Chromatin

During cell division, correct positioning of chromosomes in mitotic and meiotic spindles depends on interactions of microtubules with kinetochores and, especially in higher eukaryotes, with the chromosome arms [1, 2]. Chromokinesins, highly concentrated on mitotic and meiotic chromatin, are thought to actively push the chromosome arms toward the spindle center, thereby contributing to chromosome alignment at the metaphase plate in early mitosis [1-9]. How many distinct classes of chromokinesins exist and how they cooperate to form a motile chromatin-microtubule interface are not known. Using a novel experimental assay with nonkinetochore chromatin reconstituted from Xenopus egg extract, we demonstrate that the microtubule motility generated on chromatin is continuous and plus-end directed. Using specific antibody depletions, we identify two distinct chromokinesins, kinesin-10 (Xkid) [8, 10, 11] and kinesin-4 (Xklp1) [12, 13], as the major activities mediating the interaction of meiotic chromatin with microtubules. Interestingly, we find that the slower motor, kinesin-10, more efficiently recruits microtubules and also dominates in collective microtubule transport both in the close-to-physiological environment of chromatin and also in a minimal in vitro assay. Our results provide an identification of the molecular activities involved in the generation of motor protein-mediated chromosome arm motility and yield mechanistic insight into the cooperation of the two major chromokinesins.

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment ("zippering") or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.

Phenomenological simulation of self-organization of microtubule driven by dynein c

It was recently noticed that in vitro motility assays, driven by random distributed dynein c, microtubules could form self-organized circular patterns, which could be of importance to the design of nanobiomechanical machines. In order to determine key parameters that control the self-organized movement of microtubules, a phenomenological modeling study taking account of the microtubule joining probability distribution and microtubule bias was conducted to investigate the self-organization of microtubules driven by dynein motors.

Effects of Confinement on the Self-Organization of Microtubules and Motors

The regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, "Y-branching" organizations, cortical-like bundles, "rackets," and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo.

Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase

Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called “interphase microtubule assemblies” (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic “plus” ends facing the cell tips and their “minus” ends overlapping at the cell middle. Although the main protein factors involved in interphase microtubule organization have been identified, an understanding of how their collective interaction with microtubules leads to the organization and structures observed in vivo is lacking. We present a physical model of microtubule dynamics that aims to provide a quantitative description of the self-organization process. First, we solve equations for the microtubule length distribution in steady-state, taking into account the way that a limited tubulin pool affects the nucleation, growth and shrinkage of microtubules. Then we incorporate passive and active crosslinkers (the bundling factor Ase1 and molecular motor Klp2) and investigate the formation of IMA structures. Analytical results are complemented by a 3D stochastic simulation.

CLASP Modulates Microtubule-Cortex Interaction during Self-Organization of Acentrosomal Microtubules

CLASP proteins associate with either the plus ends or sidewalls of microtubules depending on the subcellular location and cell type. In plant cells, CLASP's distribution along the full length of microtubules corresponds with the uniform anchorage of microtubules to the cell cortex. Using live cell imaging, we show here that loss of CLASP in Arabidopsis thaliana results in partial detachment of microtubules from the cortex. The detached portions undergo extensive waving, distortion, and changes in orientation, particularly when exposed to the forces of cytoplasmic streaming. These deviations from the normal linear polymerization trajectories increase the likelihood of intermicrotubule encounters that are favorable for subsequent bundle formation. Consistent with this, cortical microtubules in clasp-1 leaf epidermal cells are hyper-parallel. On the basis of these data, we identify a novel mechanism where modulation of CLASP activity governs microtubule-cortex attachment, thereby contributing to self-organization of cortical microtubules.

Simulation studies of self-organization of microtubules and molecular motors

We perform Monte Carlo type simulation studies of self-organization of microtubules interacting with molecular motors. We model microtubules as stiff polar rods of equal length exhibiting anisotropic diffusion in the plane. The molecular motors are implicitly introduced by specifying certain probabilistic collision rules resulting in realignment of the rods. This approximation of the complicated microtubule-motor interaction by a simple instant collision allows us to bypass the "computational bottlenecks" associated with the details of the diffusion and the dynamics of motors and the reorientation of microtubules. Consequently, we are able to perform simulations of large ensembles of microtubules and motors on a very large time scale. This simple model reproduces all important phenomenology observed in in vitro experiments: Formation of vortices for low motor density and raylike asters and bundles for higher motor density.

The chirality of ciliary beats

Many eukaryotic cells possess cilia which are motile, whip-like appendages that can oscillate and thereby induce motion and fluid flows. These organelles contain a highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of nine doublets of microtubules. Complex bending waves emerge from the interplay of active internal forces generated by dynein motor proteins within the structure. These bending waves are typically chiral and often exhibit a sense of rotation. In order to study how the shape of the beat emerges from the axonemal structure, we present a three-dimensional description of ciliary dynamics based on the self-organization of dynein motors and microtubules. Taking into account both bending and twisting of the cilium, we determine self-organized beating patterns and find that modes with both a clockwise and anticlockwise sense of rotation exist. Because of the axonemal chirality, only one of these modes is selected dynamically for given parameter values and properties of dynein motors. This physical mechanism, which underlies the selection of a beating pattern with specific sense of rotation, triggers the breaking of the left–right symmetry of developing embryos which is induced by asymmetric fluid flows that are generated by rotating cilia.

Bundling, sliding, and pulling microtubules in cells and in silico

Microtubules and other proteins self-organize into complex dynamic structures such as the mitotic spindle, which separates the chromosomes during cell division. Much is known about the individual molecular players involved in assembly and positioning of the mitotic spindle, but how they act together to generate the often unexpected behavior of the whole microtubule system is not understood. Two recent papers use a combination of experimental (imaging) and theoretical (computer simulation) methods to explore the formation of bipolar linear microtubule arrays in fission yeast and the oscillatory movement of the mitotic spindle in the nematode worm. In the simulation approach, the rules for the interactions of the components (microtubules and microtubule-associated proteins) are specified and the evolution of the system is followed, with the aim of identifying the minimal set of components that can mimic the real system. The work on fission yeast concludes that bipolar microtubule structures can arise from self-organization of microtubules through nucleators, bundlers, and sliders, without a requirement for a special microtubule-organizing center. The work on the worm embryo suggests that both the positive feedback that drives oscillations and the centering force that limits their amplitude may arise from microtubule pulling forces. The systems approach exemplified by these papers should stimulate new experiments aimed at discovering the principles of cellular organization.

Retraction Note: Nematic Ordering Pattern Formation in the Process of Self-Organization of Microtubules in a Gravitational Field.

[This retracts the article DOI: 10.1007/s10867-006-9032-x.].

Mechanisms of Spindle-Pole Organization Are Influenced by Kinetochore Activity in Mammalian Cells

The spindle is a fusiform bipolar-microtubule array that is responsible for chromosome segregation during mitosis. Focused poles are an essential feature of spindles in vertebrate somatic cells, and pole focusing has been shown to occur through a centrosome-independent self-organization mechanism where microtubule motors cross-link and focus microtubule minus ends. Most of our understanding of this mechanism for pole focusing derives from studies performed in cell-free extracts devoid of centrosomes and kinetochores. Here, we examine how sustained force from kinetochores influences the mechanism of pole focusing in cultured cells. We show that the motor-driven self-organization activities associated with NuMA (i.e., cytoplasmic dynein) and HSET are not necessary for pole focusing if sustained force from kinetochores is inhibited in Nuf2- or Mis12-deficient cells. Instead, pole organization relies on TPX2 as it cross-links spindle microtubules to centrosome-associated mitotic asters. Thus, both motor-driven and static-cross-linking mechanisms contribute to spindle-pole organization, and kinetochore activity influences the mechanism of spindle-pole organization. The motor-driven self-organization of microtubule minus ends at spindle poles is needed to organize spindle poles in vertebrate somatic cells when kinetochores actively exert force on spindle microtubules.

Nematic ordering pattern formation in the process of self-organization of microtubules in a gravitational field.

Papaseit et al. (Proc. Natl. Acad. Sci. U.S.A. 97, 8364, 2000) showed the decisive role of gravity in the formation of patterns by assemblies of microtubules in vitro. By virtue of a functional scaling, the free energy for MT systems in a gravitational field was constructed. The influence of the gravitational field on MT's self-organization process, that can lead to the isotropic to nematic phase transition, is the focus of this paper. A coupling of a concentration gradient with orientational order characteristic of nematic ordering pattern formation is the new feature emerging in the presence of gravity. The concentration range corresponding to a phase coexistence region increases with increasing g or MT concentration. Gravity facilitates the isotropic to nematic phase transition leading to a significantly broader transition region. The phase transition represents the interplay between the growth in the isotropic phase and the precipitation into the nematic phase. We also present and discuss the numerical results obtained for local MT concentration change with the height of the vessel, order parameter and phase transition properties.

Dynein and dynactin as organizers of the system of cell microtubules

A review of the role of the microtubule motor dynein and its cofactor dynactin in the formation of a radial system of microtubules in the interphase cells and of mitotic spindle. Deciphering of the structure, functions, and regulation of activity of dynein and dynactin promoted the understanding of mechanisms of cell and tissue morphogenesis, since it turned out that these cells help the cell in finding its center and organize microtubule-determined anisotropy of intracellular space. The structure of dynein and dynactin molecules has been considered, as well as possible pathways of regulation of the dynein activity and the role of dynein in transport of cell components along the microtubules. Attention has also been paid to the functions of dynein and dynactin not related directly to transport: their involvement in the formation of an interphase radial system of microtubules. This system can be formed by self-organization of microtubules and dynein-containing organelles or via organization of microtubules by the centrosome, whose functioning requires dynein. In addition, dynein and dynactin are responsible for cell polarization during its movement, as well as for the position of nucleus, centrosomes, and mitotic spindle in the cell.