Microtubule Bundles Research Articles

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.

Geometrical nonlinear elasticity of axon under tension: A coarse-grained computational study

Axon bundles cross-linked by microtubule (MT) associate proteins and bounded by a shell skeleton are critical for normal function of neurons. Understanding effects of the complexly geometrical parameters on their mechanical properties can help gain a biomechanical perspective on the neurological functions of axons and thus brain disorders caused by the structural failure of axons. Here, the tensile mechanical properties of MT bundles cross-linked by tau proteins are investigated by systematically tuning MT length, axonal cross-section radius, and tau protein spacing in a bead-spring coarse-grained model. Our results indicate that the stress-strain curves of axons can be divided into two regimes, a nonlinear elastic regime dominated by rigid-body like inter-MT sliding, and a linear elastic regime dominated by affine deformation of both tau proteins and MTs. From the energetic analyses, first, the tau proteins dominate the mechanical performance of axons under tension. In the nonlinear regime, tau proteins undergo a rigid-body like rotating motion rather than elongating, whereas in the nonlinear elastic regime, tau proteins undergo a flexible elongating deformation along the MT axis. Second, as the average spacing between adjacent tau proteins along the MT axial direction increases from 25 to 125 nm, the Young’s modulus of axon experiences a linear decrease whereas with the average space varying from 125 to 175 nm, and later reaches a plateau value with a stable fluctuation. Third, the increment of the cross-section radius of the MT bundle leads to a decrease in Young’s modulus of axon, which is possibly attributed to the decrease in MT numbers per cross section. Overall, our research findings offer a new perspective into understanding the effects of geometrical parameters on the mechanics of MT bundles as well as serving as a theoretical basis for the development of artificial MT complexes potentially toward medical applications.

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons.

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.

Nuclear Lamin A/C Expression Is a Key Determinant of Paclitaxel Sensitivity

Paclitaxel is a key member of the Taxane (paclitaxel [originally named taxol], docetaxel/Taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anticancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from lamin A/C-null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wild-type cells. Forced overexpression of lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved by either the withdrawal of serum or the addition of mitotic inhibitors. These results provide a new understanding for the mitotis-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear lamin A/C protein levels correlate with nuclear shape deformation and are a key determinant of paclitaxel sensitivity of cancer cells.

Microtubule associated protein WAVE DAMPENED2-LIKE (WDL) controls microtubule bundling and the stability of the site of tip-growth in Marchantia polymorpha rhizoids.

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes–WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)–are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.

An axonemal alteration in apical endometria of human adenomyosis.

Is there any change in the distribution of microvilli and microtubules in the apical endometria of women with adenomyosis? We observed microvilli damage in the apical endometria and an axonemal alteration characterized by abnormal distribution of longitudinal bundles of microtubules within microvilli in women with adenomyosis. Human adenomyosis has a negative impact on female fertility. Abnormal utero-tubal sperm transport, tissue inflammation and toxic effect of chemical mediators have been proposed as contributing factors. Inflammation-induced damage of mucosal cilia in the Fallopian tube has been reported. However, information on inflammation-induced damage of microvilli on the apical endometrial cells and its core bundles of microtubules in adenomyosis remains unknown. This is a prospective cohort study with subjects undergoing laparoscopic surgery or hysterectomy for clinical indication and evaluations of endometrial biopsy samples in two academic university hospitals. During the period between March 2015 and December 2018, endometrial biopsy samples were prospectively collected from 15 control women and 45 women with adenomyosis for immunohistochemical analysis and a separate cohort of 10 control women with cervical intraepithelial neoplasia Grade 3 (CIN3) and 20 women with adenomyosis for analysis by immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemical study, endometrial biopsy samples were prospectively collected from 15 control women with fibroids, 25 women with focal adenomyosis and 20 women with diffuse adenomyosis after surgery. The diagnosis of fibroid and adenomyosis was made clinically by transvaginal ultrasonography and magnetic resonance imaging and confirmed by histology. Immunohistochemical analysis was performed retrospectively using antibody against CD68 (marker of macrophages) in endometrial biopsy specimens of women with and without adenomyosis. TEM was performed with the apical endometria collected from a separate cohort of 10 control women with CIN3 and 20 women with focal and diffuse adenomyosis for the identification of any change in the distribution of microvilli and longitudinal bundles of microtubules within microvilli. Comparing to control endometria and contralateral side, tissue infiltration of macrophages (Mφ) in the endometria was significantly higher on the ipsilateral side of focal adenomyosis (P = 0.02 and P = 0.03, respectively) and anterior/posterior walls of diffuse adenomyosis (P = 0.01 for both). In a subgroup analysis of patients with focal adenomyosis with and without symptoms, the endometria of symptomatic women displayed a tendency of higher Mφ infiltration on the ipsilateral side than in asymptomatic women (P = 0.07). Comparing to contralateral side endometria of symptomatic women, Mφ infiltration was significantly higher in the endometria of symptomatic women collected from the ipsilateral side of focal adenomyosis (P = 0.03). We found a significantly less tissue infiltration of Mφ in the endometria of women with CIN3 than that in endometria of women with focal adenomyosis. TEM analysis showed that number of microvilli in the endometria was significantly decreased on the ipsilateral side (P = 0.003) comparing to that on the contralateral side of focal adenomyosis. The Chi-squared test indicated that cases with abnormal (disruption in the normal arrangement of 9 peripheral pairs + 1 central pair) microtubules (MT) were significantly higher in women with adenomyosis than in cases with normal patterns (P = 0.0016). While contralateral side displayed significantly less abnormal MT (P = 0.0002), ipsilateral side of focal adenomyosis showed significantly higher abnormal MT (P = 0.0164) comparing to normal patterns. Cases with symptomatic adenomyosis showed significantly higher abnormal MT than normal MT (P = 0.0004). An axonemal alteration characterized by abnormal structural distribution of microtubules within microvilli in the apical endometria in response to endometrial inflammation may be involved in adverse reproductive outcome in women with adenomyosis. The average age of women in this study was high that may be associated with overall decline in fertility regardless of the presence or absence of adenomyosis or endometriosis. We collected endometrial biopsy samples from two completely separate cohorts of women for analysis by immunohiostochemistry and TEM. We need future follow-up study with increased sample size and from the same patients to precisely clarify the mechanistic link between axonemal alteration and negative fertility outcome. Our current findings may have some biological implication to better understand the endometrial epithelial biology and pathology in women with adenomyosis and may open the avenue for future study in other reproductive diseases. The ultra-structural abnormalities of microvilli and microtubules in the apical endometria in response to tissue inflammatory reaction may clarify the possible association between negative fertility outcome and adenomyosis. Our findings may be clinically useful during counseling with symptomatic patients with adenomyosis desiring pregnancy. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study. N/A.

Solitons in nerve axons

An axon or nerve fiber is a long, slender projection of a nerve cell, or neuron, in vertebrates that typically conducts electrical impulse known as action potentials away from the nerve cell body. Axons are the cable-like protrusions of neurons which wire up the nervous system. These delicate structures frequently need to survive for a lifetime. Parallel polar, bundles of microtubules run beside axons to form their structural backbones and are acting as highways for life-sustaining transportation. In this paper, we investigate the excitations of soliton along axons that are governed by the Nerve Soliton (NS) model. The NS model of nerve axon is governed by the discrete nonlinear equation of motion derived using the finite difference method. We done modulational instability (MI) analysis for the discrete equation of motion with effect of damping force by using Rotating wave approximation (RWA). The inherent intrinsic localized wave modes were generated which is responsible for energy localization in the nerve axons.

First spermatological data on the digenean genus Ityogonimus derived from the description of sperm characters of Ityogonimus ocreatus (Brachylaimidae: Ityogoniminae)

The present study describes the ultrastructural organization of the spermatozoa of the brachylaimid digenean Ityogonimus ocreatus (Ityogoniminae) by means of transmission electron microscopy (TEM). Live digeneans were collected from the digestive tract of an Iberian mole Talpa occidentalis (Eulipotyphla, Talpidae) captured accidentally during a vole pest control campaign in Priesca (Asturias, Spain). The TEM study reveals that the I. ocreatus sperm are filiform, tapered at both extremities, and have two 9+'1' trepaxonematan axonemes, external ornamentation of the plasma membrane associated with cortical microtubules, spine-like bodies, two bundles of parallel cortical microtubules and one mitochondrion overlapping the anterior part of the nucleus. The external ornamentation of the plasma membrane is located in the posterior part of the anterior region. The maximum number of parallel cortical microtubules (45) is located in the anterior part of the sperm cells. Our results are compared with the available data on the family Brachylaimidae, especially on the other Ityogoniminae studied to date (Scaphiostomum palaearcticum).

Centrosome‐mediated microtubule remodeling during axon formation in human iPSC‐derived neurons

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule‐organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human‐induced pluripotent stem cell (iPSC)‐derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule‐associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live‐cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus‐end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC‐derived neurons, thereby laying the foundation for further axon development and function.

Dynein self-organizes while translocating the centrosome in T-cells.

T-cells massively restructure their internal architecture upon reaching an antigen-presenting cell (APC) to form the immunological synapse (IS), a cell–cell interface necessary for efficient elimination of the APC. This reorganization occurs through tight coordination of cytoskeletal processes: actin forms a peripheral ring, and dynein motors translocate the centrosome toward the IS. A recent study proposed that centrosome translocation involves a microtubule (MT) bundle that connects the centrosome perpendicularly to dynein at the synapse center: the “stalk.” The synapse center, however, is actin-depleted, while actin was assumed to anchor dynein. We propose that dynein is attached to mobile membrane anchors, and investigate this model with computer simulations. We find that dynein organizes into a cluster in the synapse when translocating the centrosome, aligning MTs into a stalk. By implementing both a MT-capture-shrinkage and a MT-sliding mechanism, we explicitly demonstrate that this organization occurs in both systems. However, results obtained with MT-sliding dynein are more robust and display a stalk morphology consistent with our experimental data obtained with expansion microscopy. Thus, our simulations suggest that actin organization in T-cells during activation defines a specific geometry in which MT-sliding dynein can self-organize into a cluster and cause stalk formation.

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon.

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.

Differential Regulation of Single Microtubules and Cross-Linked Bundles by a Minimal Three-Protein Module

A remarkable feature of the microtubule cytoskeleton is co-existence of distinct populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: the microtubule-cross-linker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of cross-linked bundles. This striking differentiation arises from (i) higher activity of CLASP1 despite lower microtubule occupancy than Kif4A, (ii) amplification of Kif4A activity on microtubule bundles through stronger PRC1-affinity. In contrast to canonical mechanisms where antagonistic regulators set microtubule lengths of single arrays, our findings illuminate design principles by which their collective activity instead creates microenvironments comprised of arrays with distinct dynamic properties.

Understanding the Mechanisms that Give Rise to the Mammalian Spindle's Response to Force using Theory and Microneedle Manipulation

The spindle is the force-generating structure that drives chromosome segregation at cell division. In mammalian spindles, bundles of spindle microtubules called kinetochore-fibers (k-fibers) pull on chromosomes to move them. While we know nearly all components necessary for spindle function, how k-fibers respond to force and maintain themselves under force remains poorly understood. Our recent ability to exert local force on the mammalian spindle with microneedles provides key information to answering this question.

Divalent Cations Induce Non-Equilibrium Phase Transitions in Tau-Mediated Microtubule Bundles Undergoing Dynamic Instability

The microtubule-associated protein tau is known for its ability to alter microtubule (MT) dynamic instability in developing and mature neurons and for its role in the pathology of many neurodegenerative diseases. Bundling of MTs into linear arrays (i.e. MT fascicles) is thought to be a cardinal feature of the axon initial segment of neurons, and tau's role in mediating MT-MT interactions in these arrays is thought to be critical for neuron function; however, the exact mechanism by which tau links and dictates the wide spacing of bundled MTs remains unclear. To help elucidate the nature of tau's role in physiological bundling of MTs, we have expanded on a recent platform for studying paclitaxel-free in vitro reaction mixtures of tubulin, GTP, and wild-type tau under dissipative, out-of-equilibrium conditions at 37 C. Using synchrotron small-angle x-ray scattering and transmission electron microscopy, we show that Ca2+ or Mg2+ in the mM concentration range can induce changes in the assembly of tubulin structures, revealing a phase transition between the previously reported widely spaced bundles and a novel transient bundled state with intermediate spacing. The latter bundled state is followed by rapid MT depolymerization and the formation of tubulin oligomers and rings. The transitions between each of these phases and their dependence on both time and cation concentration will be discussed.

Static adhesion hysteresis in elastic structures

Adhesive interactions between elastic structures such as graphene sheets, carbon nanotubes, and microtubules have been shown to exhibit hysteresis due to irrecoverable energy loss associated with bond breakage, even in static (rate-independent) experiments. To understand this phenomenon, we start with a minimal theory for the peeling of a thin sheet from a substrate, coupling the local event of bond breaking to the nonlocal elastic relaxation of the sheet and show that this can drive static adhesion hysteresis over a bonding/debonding cycle. Using this model we quantify hysteresis in terms of the adhesion and elasticity parameters of the system. This allows us to derive a scaling relation that preserves hysteresis at different levels of granularity while resolving a seeming paradox of lattice trapping in the continuum limit of a discrete fracture process. Finally, to verify our theory, we use new experiments to demonstrate and measure adhesion hysteresis in bundled microtubules.

Machine learning forecasting of active nematics.

Active nematics are a class of far-from-equilibrium materials characterized by local orientational order of force-generating, anisotropic constitutes. Traditional methods for predicting the dynamics of active nematics rely on hydrodynamic models, which accurately describe idealized flows and many of the steady-state properties, but do not capture certain detailed dynamics of experimental active nematics. We have developed a deep learning approach that uses a Convolutional Long-Short-Term-Memory (ConvLSTM) algorithm to automatically learn and forecast the dynamics of active nematics. We demonstrate our purely data-driven approach on experiments of 2D unconfined active nematics of extensile microtubule bundles, as well as on data from numerical simulations of active nematics.

Live imaging of microtubule organization, cell expansion, and intercellular space formation in Arabidopsis leaf spongy mesophyll cells.

Leaf spongy mesophyll cells form an interconnected network of branched cells and intercellular spaces to maximize the surface area available for light capture and photosynthetic gas exchange. To investigate the morphogenetic events leading to cell separation and branching in Arabidopsis thaliana, we used mesophyll-specific promoters to facilitate imaging of mesophyll cell shape and microtubule (MT) organization over multiple spatiotemporal scales without interference from the overlying epidermal cells. We show that cells enlarge by selective expansion of cell wall regions in contact with intercellular spaces. Cell-cell contacts remain relatively fixed in size, forming the termini of interconnecting branches. Surprisingly, classic schizogeny (de-adhesion of neighboring cells) is relatively infrequent, being related to the local topology of cell junctions during early expansion. Intercellular spaces cue the position of stable MT bundles, which in turn promote efficient dilation of intercellular spaces and cell branching. Our data provide insights into mesophyll morphogenesis and MT organization and lay the groundwork for future investigations.

Confinement Controls the Bend Instability of Three-Dimensional Active Liquid Crystals.

Spontaneous growth of long-wavelength deformations is a defining feature of active liquid crystals. We investigate the effect of confinement on the instability of 3D active liquid crystals in the isotropic phase composed of extensile microtubule bundles and kinesin molecular motors. When shear aligned, such fluids exhibit finite-wavelength self-amplifying bend deformations. By systematically changing the channel size we elucidate how the instability wavelength and its growth rate depend on the channel dimensions. Experimental findings are qualitatively consistent with a minimal hydrodynamic model, where the fastest growing deformation is set by a balance of active driving and elastic relaxation. Our results demonstrate that confinement determines the structure and dynamics of active fluids on all experimentally accessible length scales.