High-throughput characterization of cortical microtubule arrays response to anisotropic tensile stress.

BACKGROUND: Plants can perceive and respond to mechanical signals. For instance, cortical microtubule (CMT) arrays usually reorganize following the predicted maximal tensile stress orientation at the cell and tissue level. While research in the last few years has started to uncover some of the mechanisms mediating these responses, much remains to be discovered, including in most cases the actual nature of the mechanosensors. Such discovery is hampered by the absence of adequate quantification tools that allow the accurate and sensitive detection of phenotypes, along with high throughput and automated handling of large datasets that can be generated with recent imaging devices. RESULTS: Here we describe an image processing workflow specifically designed to quantify CMT arrays response to tensile stress in time-lapse datasets following an ablation in the epidermis - a simple and robust method to change mechanical stress pattern. Our Fiji-based workflow puts together several plugins and algorithms under the form of user-friendly macros that automate the analysis process and remove user bias in the quantification. One of the key aspects is also the implementation of a simple geometry-based proxy to estimate stress patterns around the ablation site and compare it with the actual CMT arrays orientation. Testing our workflow on well-established reporter lines and mutants revealed subtle differences in the response over time, as well as the possibility to uncouple the anisotropic and orientational response. CONCLUSION: This new workflow opens the way to dissect with unprecedented detail the mechanisms controlling microtubule arrays re-organization, and potentially uncover the still largely elusive plant mechanosensors.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Dynamic measurement of cytosolic pH and [NO3-] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis.

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thaliana Chloride Channel a) is a vacuolar NO3-/H+ exchanger regulating stomata aperture in Athaliana Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo in Arabidopsis guard cells. We first found that ClopHensor is not only a Cl- but also, an NO3- sensor. We were then able to quantify the variations of NO3- and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3- In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3- and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.

Polar expedition: mechanisms for protein polar localization.

Most cells show asymmetry in their shape or in the organization of their components that results in poles with different properties. This is a fundamental feature that participates in modulating the development of an organism and its responses to external stimuli. In plants, a number of proteins that are important for developmental and physiological processes have been shown to display polar localization. However, how these polarities are established, maintained, or dynamically modulated is still largely unclear for most of these proteins. In this review we report recent updates on the mechanisms of polar protein localization, focusing on a subset of these proteins that are the focus of current research efforts.

Cav3.2 T-type calcium channels shape electrical firing in mouse Lamina II neurons.

The T-type calcium channel, Cav3.2, is necessary for acute pain perception, as well as mechanical and cold allodynia in mice. Being found throughout sensory pathways, from excitatory primary afferent neurons up to pain matrix structures, it is a promising target for analgesics. In our study, Cav3.2 was detected in ~60% of the lamina II (LII) neurons of the spinal cord, a site for integration of sensory processing. It was co-expressed with Tlx3 and Pax2, markers of excitatory and inhibitory interneurons, as well as nNOS, calretinin, calbindin, PKCγ and not parvalbumin. Non-selective T-type channel blockers slowed the inhibitory but not the excitatory transmission in LII neurons. Furthermore, T-type channel blockers modified the intrinsic properties of LII neurons, abolishing low-threshold activated currents, rebound depolarizations, and blunting excitability. The recording of Cav3.2-positive LII neurons, after intraspinal injection of AAV-DJ-Cav3.2-mcherry, showed that their intrinsic properties resembled those of the global population. However, Cav3.2 ablation in the dorsal horn of Cav3.2GFP-Flox KI mice after intraspinal injection of AAV-DJ-Cav3.2-Cre-IRES-mcherry, had drastic effects. Indeed, it (1) blunted the likelihood of transient firing patterns; (2) blunted the likelihood and the amplitude of rebound depolarizations, (3) eliminated action potential pairing, and (4) remodeled the kinetics of the action potentials. In contrast, the properties of Cav3.2-positive neurons were only marginally modified in Cav3.1 knockout mice. Overall, in addition to their previously established roles in the superficial spinal cord and in primary afferent neurons, Cav3.2 channel appear to be necessary for specific, significant and multiple controls of LII neuron excitability.