Tip-growing cells of, amongst others, plants and fungi secrete wall materials in a highly polarized fashion for fast and efficient colonization of the environment. A polarized microtubule cytoskeleton, in which most microtubule ends are directed toward the growing apex, has been implicated in directing growth. Its organizing principles, in particular regarding maintenance of network unipolarity, have remained elusive. We show that a kinesin-4 protein, hitherto best known for a role in cytokinesis, suppresses encounters between antiparallel microtubules. Without this activity, microtubules hyper-aligned along the growth axis and increasingly grew away from the apex. Cells themselves displayed an overly straight growth path and a delayed gravitropic response. This result revealed conflicting systemic needs for a stable growth direction and an ability to change course in response to extracellular cues. Thus, the use of selective inhibition of microtubule growth at antiparallel overlaps constitutes a new organizing principle within a unipolar microtubule array.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans -differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

Cells can position multiple copies of components like carboxysomes, nucleoids, and nuclei at regular intervals. By controlling positions, cells, for example, ensure equal partitioning of organelles over daughter cells and, in the case of nuclei, control cell sizes during cellularization. Mechanisms that generate regular patterns are as yet poorly understood. We used fission yeast cell cycle mutants to investigate the dispersion of multiple nuclei by microtubule-generated forces in single cells. After removing internuclear attractive forces by microtubule-based molecular motors, we observed the establishment of regular patterns of nuclei. Based on live-cell imaging, we hypothesized that microtubule growth within internuclear spaces pushes neighbouring nuclei apart. In the proposed mechanism, which was validated by stochastic simulations, the repulsive force weakens with increasing separation because stochastic shortening events limit the extent over which microtubules generate forces. Our results, therefore, demonstrate how cells can exploit the dynamics of microtubule growth for the equidistant positioning of organelles.

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit-ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. We established long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to investigate the microtubule re-organization. The initial disperse microtubule array rapidly readjusted into well-defined microtubule bands, which required local de-stabilization of individual microtubules in band-interspersing gap regions. Using extensive microtubule simulations, we could recapitulate the process in silico and found that local recruitment of microtubule-bound nucleation is critical for pattern formation, which we confirmed in vivo . Our simulations further indicated that the initial microtubule alignment impact microtubule band patterning. We confirmed this prediction using katanin mutants, which have microtubule organization defects, and uncovered active KATANIN recruitment to the forming microtubule bands. Our combination of quantitative microscopy and modelling outlines a framework towards a comprehensive understanding of microtubule re-organization during wall pattern formation.

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

Different from animal cells that divide by constriction of the cortex inward, cells of land plants divide by initiating a new cell-wall segment from their center. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands toward the parental cell wall [1-3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4-6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7-9]. How vesicle immobilization and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell-plate formation is lacking. Here we show that membranous material for cell-plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4-mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell-plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell-wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10, 11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end-to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8 Delta tail). In contrast to wild-type Cin8, the motility of single molecules of Cin8 Delta tail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8 Delta tail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8 Delta tail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus-and minus-end-directed motility.

Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks.

The walls of microtubules can self-repair bending-induced damage.

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants.

Polymerization ratchets formed by the assembly of actin filaments and microtubules are possibly the simplest realizations of biological thermal ratchets. A variety of experimental evidence exists that significant forces are generated by these processes, but quantitative studies lag far behind similar studies for molecular motors such as kinesin and myosin. Here we present a discussion of the theory of polymerization ratchets as well as experimental techniques used in our laboratory for the study of forces generated by single growing microtubules. Data obtained with these techniques provide us with valuable information that may eventually allow us to distinguish between different models for the growth of microtubules.

MAP65s function as microtubule cross-linking proteins, and three of five P. patens MAP65s localize to the phragmoplast equator in protonemal cells. Simultaneous RNAi-mediated knockdown of those three MAP65s compromised microtubule bipolarity and affected accumulation and/or fusion of cell plate–destined vesicles at the equatorial plane. The phragmoplast, a plant-specific apparatus that mediates cytokinesis, mainly consists of microtubules (MTs) arranged in a bipolar fashion, such that their plus ends interdigitate at the equator. Membrane vesicles are thought to move along the MTs toward the equator and fuse to form the cell plate. Although several genes required for phragmoplast MT organization have been identified, the mechanisms that maintain the bipolarity of phragmoplasts remain poorly understood. Here, we show that engaging phragmoplast MTs in a bipolar fashion in protonemal cells of the moss Physcomitrella patens requires the conserved MT cross-linking protein MICROTUBULE-ASSOCIATED PROTEIN65 (MAP65). Simultaneous knockdown of the three MAP65s expressed in those cells severely compromised MT interdigitation at the phragmoplast equator after anaphase onset, resulting in the collapse of the phragmoplast in telophase. Cytokinetic vesicles initially localized to the anaphase midzone as normal but failed to further accumulate in the next several minutes, although the bipolarity of the MT array was preserved. Our data indicate that the presence of bipolar MT arrays is insufficient for vesicle accumulation at the equator and further suggest that MAP65-mediated MT interdigitation is a prerequisite for maintenance of bipolarity of the phragmoplast and accumulation and/or fusion of cell plate–destined vesicles at the equatorial plane.

Overlapping antiparallel microtubules are important in cellular structures such as the mitotic spindle. Diez and colleagues use an in vitro system and mathematical modelling to show that the formation of stable overlaps involves a motor such as kinesin-14, which slides microtubules apart, and a passive microtubule crosslinker, Ase1, which accumulates at microtubule overlapping regions and slows microtubule sliding to prevent their separation.

Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.

Sets of overlapping microtubules support the segregation of chromosomes by linking the poles of mitotic spindles. Recent work examines the effect of putting these linkages under pressure by the activation of dicentric chromosomes and sheds new light on the structural role of several well-known spindle midzone proteins.