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MAP65-1/Ase1 decrease the flexural rigidity of individual MTs. (A) Measurement of MT persistence length. (a) Experimental setup. MT seeds are introduced in a flowthrough chamber composed of a micropattern slide (bar shape) saturated with NeutrAvidin and a glass support. They are aligned on functionalized bar patterns by the flow and attached on the micropattern surface via biotin–NeutrAvidin link (step 1). MT seeds are further elongated by the addition of Alexa-labeled tubulin in the presence or absence of MAP65 and in the presence of fluorescent beads (step 2). When MTs reach a length of 10 μm on average, the elongation mix is perfused into the flow chamber perpendicular to the elongating MTs in order to bend them (step 3). When the flow speed reaches its maximum and when it is stabilized, MT bending is measured (step 4). (b) Time series of bending MTs that elongate in the absence or presence of 100 nM MAP65-1/Ase1. MTs are in green; MT seeds and beads are in red. (c) Superposition of the images in (b), showing the amplitude of the MT bending (red arrows). (B) Histograms of the ratio between the L p of single MTs grown in the absence of MAPs and the L p of MTs grown in the presence of 100 nM MAP65-1 or Ase1. MAP65-1 and Ase1 significantly decrease MT L p . (C) Plot of the MT L p in the presence of different concentrations of MAP65-1 (1–100 nM).
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Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. While they are highly rigid polymers with a persistence length of 1-8 mm, they may exhibit curved shape at a scale of few micrometers within cells depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poor...
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... 3: MT bundle flexibility is differently regulated, depending on MAP65 cross-linker. (A) Experimental setup (top). MT seed bundles are immobilized on functionalized bar-shape patterns via biotin–NeutrAvidin link (step 1), and further elongated by perfusing tubulin in presence of MAP65 and fluorescent beads as described in Figure 1 (step 2). The hydrodynamic flow is applied when growing bundles have an average length between 10 and 20 μm (middle pattern; step 3). Time series of a bending bundle that elongates in the presence of tubulin, 100 nM MAP65-4, and fluorescent beads (bottom, left). MTs are in green; MT seeds and beads are in red. Line scan of the MT bundle (bottom, right). MT number in the bundle is determined by the level of fluorescence (right). (B) (a) Time series of bending bundles that elongate in the presence of tubulin and 100 nM MAP65-1. (b) Superposition of images showing the amplitude of the bending (red arrows).
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Citations
... Among them, MAP65 has the most members and is a plantspecific MAP [13]. MAP65 proteins are typically localized to one or more microtubule arrays, where they act as microtubule-binding proteins that cross-link with microtubules and promote the formation of microtubule bundles [14]. MAP65s were first discovered in tobacco (Nicotiana tabacum) [15]. ...
Microtubule-associated proteins (MAPs) play a pivotal role in the assembly and stabilization of microtubules, which are essential for plant cell growth, development, and morphogenesis. A class of plant-specific MAPs, MAP65, plays largely unexplored roles in moso bamboo (Phyllostachys edulis). This study identified 19 PeMAP65 genes in moso bamboo, systematically examining their phylogenetic relationships, conserved motifs, gene structures, collinearity, and cis-acting elements. Analysis of gene expression indicated that PeMAP65s exhibit tissue-specific expression patterns. Functional differentiation was investigated among the members of different PeMAP65 subfamilies according to their expression patterns in different development stages of bamboo shoots. The expression of PeMAP65-18 was positively correlated with the expression of genes involved in secondary cell wall (SCW) biosynthesis. Y1H and Dual-LUC assays demonstrated that the transcription of PeMAP65-18 was upregulated by PeMYB46, a key transcription factor of SCW biosynthesis. The result of subcellular localization showed that PeMAP65-18 was located in cortical microtubules. We speculate that PeMAP65-18 may play a crucial role in the SCW deposition of moso bamboo. This comprehensive analysis of the MAP65 family offers novel insights into the roles of PeMAP65s in moso bamboo, particularly in relation to the formation of SCWs.
... This excludes the possibility that microtubules incorporate MAP70-5 into their lattice or lumen during polymerization like MAP6. Indeed, several MAPs and posttranslational modifications of tubulin have been found to alter the stiffness of microtubules [25][26][27] , such as MAP65, which is capable of crosslinking microtubules 26 . It seems that the microtubule-bending by MAP70-5 relies on its capability of (1) reducing the stiffness of microtubules, (2) crosslinking microtubules, and (3) diffusive interaction with microtubules. ...
... This excludes the possibility that microtubules incorporate MAP70-5 into their lattice or lumen during polymerization like MAP6. Indeed, several MAPs and posttranslational modifications of tubulin have been found to alter the stiffness of microtubules [25][26][27] , such as MAP65, which is capable of crosslinking microtubules 26 . It seems that the microtubule-bending by MAP70-5 relies on its capability of (1) reducing the stiffness of microtubules, (2) crosslinking microtubules, and (3) diffusive interaction with microtubules. ...
Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.
... Cold signaling is integrated by the microtubule receptor system and amplified by COLD1-dependent signaling pathways [26]. Some members of the MAP65 family have been shown to reduce the stiffness of microtubules, directly affecting the force transmission of membrane rigidification, possibly by increasing the abundance of unsaturated fatty acids and adaptive reflow of the membrane by microtubules related [27]. ...
... Although microtubules do not transduce cold signals, they play a role in induce cold hardening [28]. Deeper research has also shown that when the membrane rigidify rapidly, the reduction in microtubule flexural rigidity (contributed by MAP65-2) prevents microtubule rupture under cold stress, while microtubule bundling is expected to improve force transmission to calcium channels as an actual sensory structure [27]. ...
Low temperature stress is one of the most important factors limiting plant growth and geographical distribution. In order to adapt to low temperature, plants have evolved strategies to acquire cold tolerance, known as, cold acclimation. Current molecular and genomic studies have reported that annual herbaceous and perennial woody plants share similar cold acclimation mechanisms. However, woody perennials also require extra resilience to survive cold winters. Thus, trees have acquired complex dynamic processes to control the development of dormancy and cold resistance, ensuring successful tolerance during the coldest winter season. In this review, we systemically described how woody plants perceive and transduce cold stress signals through a series of physiological changes such as calcium signaling, membrane lipid, and antioxidant changes altering downstream gene expression and epigenetic modification, ultimately bud dormancy. We extended the discussion and reviewed the processes endogenous phytohormones play in regulating the cold stress. We believe that this review will aid in the comprehension of underlying mechanisms in plant acclimation to cold stress.
... Since the bending mechanics of microtubules is well characterized (Gittes et al., 1993), many modeling studies have used shape to infer forces exerted on microtubules. This approach has been applied to single microtubules (Gittes et al., 1996;Brangwynne et al., 2006), microtubule bundles (Gadêlha et al., 2013;Portran et al., 2013), as well as k-fibers in the spindle (Rubinstein et al., 2009;Kajtez et al., 2016). To date, k-fiber models used native shapes (in unperturbed spindles) to infer underlying spindle forces, without focusing on k-fiber anchorage. ...
During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 μm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.
... It remains possible, however, that CLASP works closely with the microtubulebundling proteins MAP65-1 and MAP65-2, which are expressed and active in the RAM (Lucas and Shaw, 2012). In vitro experiments have indicated that MAP65-1 and MAP65-2 increase the flexibility of both independent and bundled microtubules, suggesting that they could mediate steep contact angle bundling (Portran et al., 2013), which occurs with TFBs. It is compelling to note that in animal and yeast cells, MAP65 orthologs directly interact with and target CLASP to regions of microtubule overlap such as the mitotic spindle (Bratman and Chang, 2007;Liu et al., 2009). ...
The transition from cell division to differentiation in primary roots is dependent on precise gradients of phytohormones, including auxin, cytokinins and brassinosteroids. The reorganization of microtubules also plays a key role in determining whether a cell will enter another round of mitosis or begin to rapidly elongate as the first step in terminal differentiation. In the last few years, progress has been made to establish connections between signaling pathways at distinct locations within the root. This review focuses on the different factors that influence whether a root cell remains in the division zone or transitions to elongation and differentiation using Arabidopsis thaliana as a model system. We highlight the role of the microtubule-associated protein CLASP as an intermediary between sustaining hormone signaling and controlling microtubule organization. We discuss new, innovative tools and methods, such as hormone sensors and computer modeling, that are allowing researchers to more accurately visualize the belowground growth dynamics of plants.
... In addition, microtubule-associated proteins and tubulin posttranslational modifications might provide nuanced control of the mechanical properties of cortical microtubules to fine-tune their force sensitivity. For example, MAP65 (Microtubule-Associated Protein of 65 kDa) proteins increase microtubule flexibility (Portran et al., 2013), whereas the mammalian MAP2 (Microtubule-Associated Protein 2) and tau proteins increase microtubule rigidity (Felgner et al., 1997). By spatially varying the microstructure of the cortical microtubule cytoskeleton, a cell could create a functionally graded sensory structure ( Figure 2A). ...
As scientists, we are at least as excited about the open questions—the things we don’t know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.
... These authors studied neuronal MAPs -Tau and MAP2 -which stabilize long neuronal structures like dendrites and axons by increasing microtubule stiffness. More recent studies have shown that other MAPs either have no effect on microtubule flexural rigidity [Cassimeris et al. 2001] or decrease it [Portran et al. 2013]. ...
Although extensively studied in vitro, the mechanics of the cytoskeleton is still largely unexplored in living cells. We use an intracellular optical tweezers-based micromanipulation technique to apply forces directly on cytoskeletal filaments in order to probe microtubules and intermediate filament mechanics and focus on how they interact mechanically. Measuring simultaneously the force applied to the filaments and their deflection, i.e. the deformation of the filaments perpendicular to their axis, as a function of time, allows us to deduce the force-deflection curves of the filaments and to characterize the rigidity of vimentin intermediate filaments and microtubules. By fitting the force-deflection curves at small forces, we show that microtubules have a lower effective stiffness than vimentin upon deflection. We then apply forces twice on the same cytoskeletal bundle to show that vimentin filaments, but not microtubules, stiffen more than three times upon repeated deflections. We further characterize the mechanical coupling between vimentin filaments and microtubules by using microtubule destabilizing and stabilizing drugs and by increasing microtubule acetylation. Interestingly, we find that these modifications do not affect the effective stiffness of vimentin filaments while destabilizing or acetylating microtubules significantly reduces vimentin filament stiffening upon repeated deflection. Altogether, these results suggest that microtubules promote stiffening of vimentin bundles under repeated mechanical stress. In sharp contrast, in cells knockout for vimentin, the mechanical properties of microtubules are unchanged. Our findings highlight the importance of the interactions between microtubules and intermediate filaments in cell mechanics and suggest that vimentin intermediate filaments are mechanosensitive structures which exhibit history-dependent mechanoresponses.
... Perfusion chambers were prepared from glass slides and coverslips using double-sided sticky tape, and the chambers were coated with silane-PEG (Creative works) following standard protocols (Portran et al., 2013). Alexa Fluor 488-phalloidin-labelled F-actin, in the absence or presence of either FH2FSI or FH2-ΔFSI, was imaged using an inverted microscope (ApoN/ TIRF 100×/1.49 ...
Dynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodelling of the cytoskeleton necessary for critical cellular processes, like axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood.
We identify Fmn2, a non-diaphanous related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodelling in growth cones. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and also in vivo. Superresolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross- bridging function of Fmn2, filopodial capture of microtubules is compromised resulting in de-stabilized filopodial protrusions and deficits in growth cone chemotaxis.
Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that modulating microtubule dynamics via associations with F-actin is central to directional motility.
... In fact, the addition of MAP65-1 to in vitrogrowing microtubules under hydrodynamic flow revealed that this protein rather make the microtubule softer. This was proposed to explain how in vivo, microtubules exhibit a lower persistence length than pure microtubules (Portran et al., 2013). ...
In plants, the development of aerial organs is indeterminate: it takes place throughout their lifespan. In contrast, the development of floral organs is determinate in Arabidopsis thaliana, each flower has the same number of floral organs. This difference in development is due to the maintenance or not of the pool of stem cells present in the stem cell niches, the meristems. During my thesis I showed that the transcriptional regulator VIP3 contributes to the regulation of the switch from indeterminate to determinate in flowers. This also revealed that the control of flower termination is not as robust as classically thought. Because VIP3 is also involved in the regulation of epigenetic marks and response to external mechanical stimuli, this work opens new questions on the role of mechanical signals in indeterminacy. On a more technical standpoint, the analysis of shoot development suffers from a lack of imaging methods with high temporal resolution and in-depth optical sectioning. During the last decade, light sheet microscopy has emerged as a competitive imaging modality in developmental biology. However, in plants, the technique has mainly been used in roots because of limits in the microscope design. During my thesis, I developed protocols allowing the imaging of aerial organs in A. thaliana using a novel light sheet set-up (Phaseview Alpha3) where shoot samples can be observed while in water. I set up an imaging pipeline from sample mounting to quantitative analysis, with a focus on local dynamics of microtubules in cotyledon epidermis in relation to cell shape. Altogether, this work provides both conceptual and technical prospects for future quantitative projects in plant development.
... Dynamic instability has not been observed in microtubules of the array, most likely due to their extensive crosslinking (Fig 1A). Some crosslinking proteins in other systems are known to promote microtubule flexibility [30], which may explain the apparent longevity of subpellicular array microtubules as they respond to forces produced by the flagellar beat. However, very little is known about the structure and function of the inter-microtubule crosslinking fibrils that organize the array or how MAPs may regulate array microtubules. ...
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.
