TY - CHAP AB - High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms. AU - Kaufmann, Walter AU - Kleindienst, David AU - Harada, Harumi AU - Shigemoto, Ryuichi ID - 9756 KW - Freeze-fracture replica: Deep learning KW - Immunogold labeling KW - Integral membrane protein KW - Electron microscopy SN - 9781071615218 T2 - Receptor and Ion Channel Detection in the Brain TI - High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL) VL - 169 ER - TY - JOUR AB - Auxin is a major plant growth regulator, but current models on auxin perception and signaling cannot explain the whole plethora of auxin effects, in particular those associated with rapid responses. A possible candidate for a component of additional auxin perception mechanisms is the AUXIN BINDING PROTEIN 1 (ABP1), whose function in planta remains unclear. Here we combined expression analysis with gain- and loss-of-function approaches to analyze the role of ABP1 in plant development. ABP1 shows a broad expression largely overlapping with, but not regulated by, transcriptional auxin response activity. Furthermore, ABP1 activity is not essential for the transcriptional auxin signaling. Genetic in planta analysis revealed that abp1 loss-of-function mutants show largely normal development with minor defects in bolting. On the other hand, ABP1 gain-of-function alleles show a broad range of growth and developmental defects, including root and hypocotyl growth and bending, lateral root and leaf development, bolting, as well as response to heat stress. At the cellular level, ABP1 gain-of-function leads to impaired auxin effect on PIN polar distribution and affects BFA-sensitive PIN intracellular aggregation. The gain-of-function analysis suggests a broad, but still mechanistically unclear involvement of ABP1 in plant development, possibly masked in abp1 loss-of-function mutants by a functional redundancy. AU - Gelová, Zuzana AU - Gallei, Michelle C AU - Pernisová, Markéta AU - Brunoud, Géraldine AU - Zhang, Xixi AU - Glanc, Matous AU - Li, Lanxin AU - Michalko, Jaroslav AU - Pavlovicova, Zlata AU - Verstraeten, Inge AU - Han, Huibin AU - Hajny, Jakub AU - Hauschild, Robert AU - Čovanová, Milada AU - Zwiewka, Marta AU - Hörmayer, Lukas AU - Fendrych, Matyas AU - Xu, Tongda AU - Vernoux, Teva AU - Friml, Jiří ID - 8931 JF - Plant Science KW - Agronomy and Crop Science KW - Plant Science KW - Genetics KW - General Medicine SN - 0168-9452 TI - Developmental roles of auxin binding protein 1 in Arabidopsis thaliana VL - 303 ER - TY - GEN AB - Growth regulation tailors plant development to its environment. A showcase is response to gravity, where shoots bend up and roots down1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots, while inhibiting it in roots via a yet unknown cellular mechanism2. Here, by combining microfluidics, live imaging, genetic engineering and phospho-proteomics in Arabidopsis thaliana, we advance our understanding how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on the rapid regulation of the apoplastic pH, a causative growth determinant. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H+-influx, causing apoplast alkalinisation. The simultaneous activation of these two counteracting mechanisms poises the root for a rapid, fine-tuned growth modulation while navigating complex soil environment. AU - Li, Lanxin AU - Verstraeten, Inge AU - Roosjen, Mark AU - Takahashi, Koji AU - Rodriguez Solovey, Lesia AU - Merrin, Jack AU - Chen, Jian AU - Shabala, Lana AU - Smet, Wouter AU - Ren, Hong AU - Vanneste, Steffen AU - Shabala, Sergey AU - De Rybel, Bert AU - Weijers, Dolf AU - Kinoshita, Toshinori AU - Gray, William M. AU - Friml, Jiří ID - 10095 SN - 2693-5015 T2 - Research Square TI - Cell surface and intracellular auxin signalling for H+-fluxes in root growth ER - TY - COMP AU - Hauschild, Robert ID - 8181 TI - Amplified centrosomes in dendritic cells promote immune cell effector functions ER - TY - COMP AB - Automated root growth analysis and tracking of root tips. AU - Hauschild, Robert ID - 8294 TI - RGtracker ER -