@article{12567, abstract = {Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.}, author = {Mrestani, Achmed and Lichter, Katharina and Sirén, Anna Leena and Heckmann, Manfred and Paul, Mila M. and Pauli, Martin}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {3}, publisher = {MDPI}, title = {{Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation}}, doi = {10.3390/ijms24032128}, volume = {24}, year = {2023}, } @article{9332, abstract = {Lateral root (LR) formation is an example of a plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE (PKL) negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PKL interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL–RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL–RBR1 is counteracted by auxin, indicating that, in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.}, author = {Ötvös, Krisztina and Miskolczi, Pál and Marhavý, Peter and Cruz-Ramírez, Alfredo and Benková, Eva and Robert, Stéphanie and Bakó, László}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {8}, publisher = {MDPI}, title = {{Pickle recruits retinoblastoma related 1 to control lateral root formation in arabidopsis}}, doi = {10.3390/ijms22083862}, volume = {22}, year = {2021}, } @article{9986, abstract = {Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan’s molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth.}, author = {Velasquez, Silvia Melina and Guo, Xiaoyuan and Gallemi, Marçal and Aryal, Bibek and Venhuizen, Peter and Barbez, Elke and Dünser, Kai Alexander and Darino, Martin and Pӗnčík, Aleš and Novák, Ondřej and Kalyna, Maria and Mouille, Gregory and Benková, Eva and Bhalerao, Rishikesh P. and Mravec, Jozef and Kleine-Vehn, Jürgen}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, keywords = {auxin, growth, cell wall, xyloglucans, hypocotyls, gravitropism}, number = {17}, publisher = {MDPI}, title = {{Xyloglucan remodeling defines auxin-dependent differential tissue expansion in plants}}, doi = {10.3390/ijms22179222}, volume = {22}, year = {2021}, } @article{6627, abstract = {Cortical microtubule arrays in elongating epidermal cells in both the root and stem of plants have the propensity of dynamic reorientations that are correlated with the activation or inhibition of growth. Factors regulating plant growth, among them the hormone auxin, have been recognized as regulators of microtubule array orientations. Some previous work in the field has aimed at elucidating the causal relationship between cell growth, the signaling of auxin or other growth-regulating factors, and microtubule array reorientations, with various conclusions. Here, we revisit this problem of causality with a comprehensive set of experiments in Arabidopsis thaliana, using the now available pharmacological and genetic tools. We use isolated, auxin-depleted hypocotyls, an experimental system allowing for full control of both growth and auxin signaling. We demonstrate that reorientation of microtubules is not directly triggered by an auxin signal during growth activation. Instead, reorientation is triggered by the activation of the growth process itself and is auxin-independent in its nature. We discuss these findings in the context of previous relevant work, including that on the mechanical regulation of microtubule array orientation.}, author = {Adamowski, Maciek and Li, Lanxin and Friml, Jiří}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {13}, publisher = {MDPI}, title = {{Reorientation of cortical microtubule arrays in the hypocotyl of arabidopsis thaliana is induced by the cell growth process and independent of auxin signaling}}, doi = {10.3390/ijms20133337}, volume = {20}, year = {2019}, } @article{14, abstract = {The intercellular transport of auxin is driven by PIN-formed (PIN) auxin efflux carriers. PINs are localized at the plasma membrane (PM) and on constitutively recycling endomembrane vesicles. Therefore, PINs can mediate auxin transport either by direct translocation across the PM or by pumping auxin into secretory vesicles (SVs), leading to its secretory release upon fusion with the PM. Which of these two mechanisms dominates is a matter of debate. Here, we addressed the issue with a mathematical modeling approach. We demonstrate that the efficiency of secretory transport depends on SV size, half-life of PINs on the PM, pH, exocytosis frequency and PIN density. 3D structured illumination microscopy (SIM) was used to determine PIN density on the PM. Combining this data with published values of the other parameters, we show that the transport activity of PINs in SVs would have to be at least 1000× greater than on the PM in order to produce a comparable macroscopic auxin transport. If both transport mechanisms operated simultaneously and PINs were equally active on SVs and PM, the contribution of secretion to the total auxin flux would be negligible. In conclusion, while secretory vesicle-mediated transport of auxin is an intriguing and theoretically possible model, it is unlikely to be a major mechanism of auxin transport inplanta.}, author = {Hille, Sander and Akhmanova, Maria and Glanc, Matous and Johnson, Alexander J and Friml, Jirí}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {11}, publisher = {MDPI}, title = {{Relative contribution of PIN-containing secretory vesicles and plasma membrane PINs to the directed auxin transport: Theoretical estimation}}, doi = {10.3390/ijms19113566}, volume = {19}, year = {2018}, }