Orientation Of Microtubules Research Articles

Single Molecule Characterization of Mitotic Kif15 Reveals Capability to Generate Force in Anti-Parallel Microtubules

Proper formation of the mitotic spindle is essential for chromosome separation and cell division. Dimeric Kinesin-12, or Kif15, has been shown to serve as a backup mechanism for cell division, promoting spindle assembly in the absence of tetrameric Kinesin-5, or Eg5. The mechanism by which Kif15 aids in the assembly of the spindle is unknown. Here, single molecule motility assays were performed using optical tweezers to evaluate the mechanistic behavior of Kif15 on microtubules (MTs). Constructs containing various sub-components of the motor were utilized to determine the role of each domain, including the motor heads, stalk, MT binding domain (N700) and the full length motor (FL). Step size, dwell times, and stall forces were measured. Rupture assays were also performed on the MT binding domain (Coil-1) to determine the binding strength and the nature of the Coil-1/MT interaction. Motility and rupture assays were repeated on subtilisin-digested MTs (dMT) in which the negatively-charged carboxyl-terminal tail of MTs, called the E-hook, is removed. We found that the Coil-1 was stronger than the stall force of the motor due to its interaction with the E-hook, revealing that Kif15 has the ability to build up mechanical strain and slide MTs apart. In order to determine the importance of MT orientation, an in vitro optical trapping and fluorescence assay was developed to determine the force generating capabilities of Kif15 in different bundle environments. We determined that Kif15 slides anti-parallel MTs apart while parallel bundles remain stationary with a small amount of antagonizing force generation. Our studies provide insight regarding how Kif15 is able to rescue bipolar spindle assembly in the absence of Eg5.

The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus.

Calcium (Ca2+) signaling and dynamic reorganization of the cytoskeleton are essential processes for the coordination and control of plant cell shape and cell growth. Calmodulin (CaM) and closely related calmodulin-like (CML) polypeptides are principal sensors of Ca2+ signals. CaM/CMLs decode and relay information encrypted by the second messenger via differential interactions with a wide spectrum of targets to modulate their diverse biochemical activities. The plant-specific IQ67 DOMAIN (IQD) family emerged as possibly the largest class of CaM-interacting proteins with undefined molecular functions and biological roles. Here, we show that the 33 members of the IQD family in Arabidopsis (Arabidopsis thaliana) differentially localize, using green fluorescent protein (GFP)-tagged proteins, to multiple and distinct subcellular sites, including microtubule (MT) arrays, plasma membrane subdomains, and nuclear compartments. Intriguingly, the various IQD-specific localization patterns coincide with the subcellular patterns of IQD-dependent recruitment of CaM, suggesting that the diverse IQD members sequester Ca2+-CaM signaling modules to specific subcellular sites for precise regulation of Ca2+-dependent processes. Because MT localization is a hallmark of most IQD family members, we quantitatively analyzed GFP-labeled MT arrays in Nicotiana benthamiana cells transiently expressing GFP-IQD fusions and observed IQD-specific MT patterns, which point to a role of IQDs in MT organization and dynamics. Indeed, stable overexpression of select IQD proteins in Arabidopsis altered cellular MT orientation, cell shape, and organ morphology. Because IQDs share biochemical properties with scaffold proteins, we propose that IQD families provide an assortment of platform proteins for integrating CaM-dependent Ca2+ signaling at multiple cellular sites to regulate cell function, shape, and growth.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Coronin 1C-free primary mouse fibroblasts exhibit robust rearrangements in the orientation of actin filaments, microtubules and intermediate filaments

Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain (“head”) of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments.

A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis

How organs reach their final shape is a central yet unresolved question in developmental biology. Here we investigate whether mechanical cues contribute to this process. We analyze the epidermal cells of the Arabidopsis sepal, focusing on cortical microtubule arrays, which align along maximal tensile stresses and restrict growth in that direction through their indirect impact on the mechanical anisotropy of cell walls. We find a good match between growth and microtubule orientation throughout most of the development of the sepal. However, at the sepal tip, where organ maturation initiates and growth slows down in later stages, microtubules remain in a configuration consistent with fast anisotropic growth, i.e., transverse, and the anisotropy of their arrays even increases. To understand this apparent paradox, we built a continuous mechanical model of a growing sepal. The model demonstrates that differential growth in the sepal can generate transverse tensile stress at the tip. Consistently, microtubules respond to mechanical perturbations and align along maximal tension at the sepal tip. Including this mechanical feedback in our growth model of the sepal, we predictan impact on sepal shape that is validated experimentally using mutants with either increased or decreased microtubule response to stress. Altogether, this suggests that a mechanical feedback loop, via microtubules acting both as stress sensor and growth regulator, channels the growth and shape of the sepal tip. We propose that this proprioception mechanism is a key step leading to growth arrest in the whole sepal in response to its own growth.

Extracting Subcellular Fibrillar Alignment with Error Estimation: Application to Microtubules

The order and orientation of cortical microtubule (CMT) arrays and their dynamics play an essential role in plant morphogenesis. To extract detailed CMT alignment structures in an objective, local, and accurate way, we propose an error-based extraction method that applies to general fluorescence intensity data on three-dimensional cell surfaces. Building on previous techniques to quantify alignments, our method can determine the statistical error for specific local regions, or the minimal scales of local regions for a desired accuracy goal. After validating our method with synthetic images with known alignments, we demonstrate the ability of our method to quantify subcellular CMT alignments on images with microtubules marked with green fluorescent protein in various cell types. Our method could also be applied to detect alignment structures in other fibrillar elements, such as actin filaments, cellulose, and collagen.

Environmental and Endogenous Control of Cortical Microtubule Orientation.

Plant growth requires a tight coordination of cell shape and anisotropic expansion. Owing to their immobility, plant cells determine body architecture through the orientation of cell division and cell expansion. Microtubule cytoskeleton represents a versatile cellular structure essential for coordinating flexible cell morphogenesis. Previous studies have identified a large number of microtubule-associated regulators that control microtubule dynamics; however, the mechanisms by which microtubule reorientation responds to exogenous and environmental stimuli are largely unknown. In this review, we describe the molecular details of microtubule dynamics that are required for cortical microtubule array pattern formation, and recapitulate current knowledge on the mechanisms by which various environmental and endogenous stimuli control cortical microtubule reorientation.

Kinesin-Cargo Motility in Random Microtubule Networks

Microtubules perform a major role in intracellular transportation, a complex and highly regulated process of motor driven transport of cargos such as organelles, protein complexes, and vesicles. Transport is essential for the survival, development, and maintenance of cells. Kinesin-1 is a major motor protein responsible for plus-end directed transport along microtubules in many cell types. Prior work in our laboratory involved studying the effect of crowding on single motors and artificial quantum-dot “cargos.” We found that single molecules were hindered in crowded conditions, but cargos were still able to move long distances. Further, we found that the orientation of multiple microtubules within bundles can alter where and how fast single motors move. Using this technique of imaging quantum-dot cargoes in a single molecule assay, we are interested to learn how single motors and cargos transport in random networks of microtubules that are bundled or crossed. We find that cargos continue to transport, but can become locally pinned to a particular location because of the local orientation and directionality of the microtubules. We are analyzing the mean squared displacements over time to determine the type of motility they display compared to multiple models of particle transport.

Dendrites In Vitro and In Vivo Contain Microtubules of Opposite Polarity and Axon Formation Correlates with Uniform Plus-End-Out Microtubule Orientation.

Live-cell imaging was used to systematically analyze microtubule organization in primary cultures of rat hippocampal neurons, dentate granule cells in mouse organotypic slices, and layer 2/3 pyramidal neuron in somatosensory cortex of living mice. In vitro and in vivo, all microtubules have a plus-end-out orientation in axons, whereas microtubules in dendrites have mixed orientations. Interestingly, newly formed neurites of nonpolarized neurons already contain mixed microtubules, and the specific organization of uniform plus-end-out microtubules only occurs during axon formation. Based on these findings, the authors propose a model in which the selective formation of uniform plus-end-out microtubules in the axon is a critical process underlying neuronal polarization.

Organization of Non-centrosomal Microtubules in Epithelial Cells.

Polarized epithelial cells contain a characteristic array of microtubules in which non-centrosomal microtubules are aligned along the apical-to-basal axis of the cell with their minus ends oriented towards the apical pole. Although this unique orientation of microtubules was discovered in the late 1980s, how this orientation is established remains unresolved partly because of limited information about molecular factors that regulate the minus ends of non-centrosomal microtubules. Recent studies, however, identified novel minus end-associated proteins, revealing mechanisms by which the polarized arrays of microtubules are established in epithelial cells. These studies have also demonstrated the importance of apico-basally orientated microtubules in intra-structural organization of cells. This review focuses on recent progress of our understanding of the molecular basis for epithelium-specific microtubule assembly and function.

Microtubule bundling plays a role in ethylene-mediated cortical microtubule reorientation in etiolated Arabidopsis hypocotyls.

The gaseous hormone ethylene is known to regulate plant growth under etiolated conditions (the 'triple response'). Although organization of cortical microtubules is essential for cell elongation, the underlying mechanisms that regulate microtubule organization by hormone signaling, including ethylene, are ambiguous. In the present study, we demonstrate that ethylene signaling participates in regulation of cortical microtubule reorientation. In particular, regulation of microtubule bundling is important for this process in etiolated hypocotyls. Time-lapse analysis indicated that selective stabilization of microtubule-bundling structures formed in various arrays is related to ethylene-mediated microtubule orientation. Bundling events and bundle growth lifetimes were significantly increased in oblique and longitudinal arrays, but decreased in transverse arrays in wild-type cells in response to ethylene. However, the effects of ethylene on microtubule bundling were partially suppressed in a microtubule-bundling protein WDL5 knockout mutant (wdl5-1). This study suggests that modulation of microtubule bundles that have formed in certain orientations plays a role in reorienting microtubule arrays in response to ethylene-mediated etiolated hypocotyl cell elongation.

CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells

Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons.

In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides 'minus-end-out' microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.

“CLASPing” tungsten's effects on microtubules with “PINs”

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.

Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress

Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism.

Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone's expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation.

Auxin inhibits expansion rate independently of cortical microtubules.

A recent publication announces that auxin inhibits expansion by a mechanism based on the orientation of cortical microtubules. This is a textbook-revising claim, but as I argue here, a claim that is supported by neither the authors' data nor previous research, and is contradicted by a simple experiment.

Patterning of Fibroblast and Matrix Anisotropy within 3D Confinement is Driven by the Cytoskeleton.

Effects of 3D confinement on cellular growth and matrix assembly are important in tissue engineering, developmental biology, and regenerative medicine. Polydimethylsiloxane wells with varying anisotropy are microfabicated using soft-lithography. Microcontact printing of bovine serum albumin is used to block cell adhesion to surfaces between wells. The orientations of fibroblast stress fibers, microtubules, and fibronectin fibrils are examined 1 day after cell seeding using laser scanning confocal microscopy, and anisotropy is quantified using a custom autocorrelation analysis. Actin, microtubules, and fibronectin exhibit higher anisotropy coefficients for cells grown in rectangular wells with aspect ratios of 1:4 and 1:8, as compared to those in wells with lower aspect ratios or in square wells. The effects of disabling individual cytoskeletal components on fibroblast responses to anisotropy are then tested by applying actin or microtubule polymerization inhibitors, Rho kinase inhibitor, or by siRNA-mediated knockdown of AXL or cofilin-1. Latrunculin A decreases cytoskeletal and matrix anisotropy, nocodazole ablates both, and Y27632 mutes cellular polarity while decreasing matrix anisotropy. AXL siRNA knockdown has little effect, as does siRNA knockdown of cofilin-1. These data identify several specific cytoskeletal strategies as targets for the manipulation of anisotropy in 3D tissue constructs.

Quantitative analysis of microtubule orientation in interdigitated leaf pavement cells

Leaf pavement cells are shaped like a jigsaw puzzle in most dicotyledon species. Molecular genetic studies have identified several genes required for pavement cells morphogenesis and proposed that microtubules play crucial roles in the interdigitation of pavement cells. In this study, we performed quantitative analysis of cortical microtubule orientation in leaf pavement cells in Arabidopsis thaliana. We captured confocal images of cortical microtubules in cotyledon leaf epidermis expressing GFP-tubulinβ and quantitatively evaluated the microtubule orientations relative to the pavement cell growth axis using original image processing techniques. Our results showed that microtubules kept parallel orientations to the growth axis during pavement cell growth. In addition, we showed that immersion treatment of seed cotyledons in solutions containing tubulin polymerization and depolymerization inhibitors decreased pavement cell complexity. Treatment with oryzalin and colchicine inhibited the symmetric division of guard mother cells.

OsKinesin-13A is an active microtubule depolymerase involved in glume length regulation via affecting cell elongation.

Grain size is an important trait influencing both the yield and quality of rice and its major determinant is glume size. However, how glume size is regulated remains largely unknown. Here, we report the characterization of OsKinesin-13A, which regulates cell elongation and glume length in rice. The mutant of OsKinesin-13A, sar1, displayed length reduction in grains and other organs including internodes, leaves and roots. The grain phenotype in sar1 was directly caused by reduction in glume length, which in turn restricted caryopsis size. Histological results revealed that length decrease in sar1 organs resulted from abnormalities in cell elongation. The orientation of cellulose microfibrils was defective in sar1. Consistently, sar1 showed reduced transverse orientation of cortical microtubules. Further observations demonstrated that microtubule turnover was decreased in sar1. OsKinesin-13A was shown to be an active microtubule depolymerase and mainly distributed on vesicles derived from the Golgi apparatus and destined for the cell surface. Thus, our results suggest that OsKinesin-13A utilizes its microtubule depolymerization activity to promote microtubule turnover, which may not only influence transverse orientation of cortical microtubules but also facilitate vesicle transport from the Golgi apparatus to the cell surface, and thus affects cellulose microfibril orientation and cell elongation.