TY - JOUR AB - The directional flow of the plant hormone auxin mediates multiple developmental processes, including patterning and tropisms. Apical and basal plasma membrane localization of AUXIN-RESISTANT1 (AUX1) and PIN-FORMED1 (PIN1) auxin transport components underpins the directionality of intercellular auxin flow in Arabidopsis thaliana roots. Here, we examined the mechanism of polar trafficking of AUX1. Real-time live cell analysis along with subcellular markers revealed that AUX1 resides at the apical plasma membrane of protophloem cells and at highly dynamic subpopulations of Golgi apparatus and endosomes in all cell types. Plasma membrane and intracellular pools of AUX1 are interconnected by actin-dependent constitutive trafficking, which is not sensitive to the vesicle trafficking inhibitor brefeldin A. AUX1 subcellular dynamics are not influenced by the auxin influx inhibitor NOA but are blocked by the auxin efflux inhibitors TIBA and PBA. Furthermore, auxin transport inhibitors and interference with the sterol composition of membranes disrupt polar AUX1 distribution at the plasma membrane. Compared with PIN1 trafficking, AUX1 dynamics display different sensitivities to trafficking inhibitors and are independent of the endosomal trafficking regulator ARF GEF GNOM. Hence, AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, providing an additional mechanism for the fine regulation of auxin transport. AU - Kleine-Vehn, Jürgen AU - Dhonukshe, Pankaj AU - Swarup, Ranjan AU - Bennett, Malcolm AU - Jirí Friml ID - 3018 IS - 11 JF - Plant Cell TI - Subcellular trafficking of the Arabidopsis auxin influx carrier AUX1 uses a novel pathway distinct from PIN1 VL - 18 ER - TY - JOUR AB - High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is ∼7 d, depending on the tissue used. AU - Hejátko, Jan AU - Blilou, Ikram AU - Brewer, Philip B AU - Jirí Friml AU - Scheres, Ben AU - Eva Benková ID - 3020 IS - 4 JF - Nature Protocols TI - In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples VL - 1 ER - TY - JOUR AB - As the field of plant molecular biology is swiftly advancing, a need has been created for methods that allow rapid and reliable in situ localization of proteins in plant cells. Here we describe a whole-mount 'immunolocalization' technique for various plant tissues, including roots, hypocotyls, cotyledons, young primary leaves and embryos of Arabidopsis thaliana and other species. The detailed protocol, recommended controls and troubleshooting are presented, along with examples of applications. The protocol consists of five main procedures: tissue fixation, tissue permeation, blocking, primary and secondary antibody incubation. Notably, the first procedure (tissue fixation) includes several steps (4-12) that are absolutely necessary for protein localization in hypocotyls, cotyledons and young primary leaves but should be omitted for other tissues. The protocol is usually done in 3 days, but could also be completed in 2 days. AU - Sauer, Michael AU - Paciorek, Tomasz AU - Eva Benková AU - Jirí Friml ID - 3015 IS - 1 JF - Nature Protocols TI - Immunocytochemical techniques for whole mount in situ protein localization in plants VL - 1 ER - TY - JOUR AB - There is a growing demand for methods that allow rapid and reliable in situ localization of proteins in plant cells. The immunocytochemistry protocol presented here can be used routinely to observe protein localization patterns in tissue sections of various plant species. This protocol is especially suitable for plant species with more-complex tissue architecture (such as maize, Zea mays), which makes it difficult to use an easier whole-mount procedure for protein localization. To facilitate the antibody-antigen reaction, it is necessary to include a wax-embedding and tissue-sectioning step. The protocol consists of the following procedures: chemical fixation of tissue, dehydration, wax embedding, sectioning, dewaxing, rehydration, blocking and antibody incubation. The detailed protocol, recommended controls and troubleshooting are presented here, along with examples of applications. AU - Paciorek, Tomasz AU - Sauer, Michael AU - Balla, Jozef AU - Wiśniewska, Justyna AU - Jirí Friml ID - 3013 IS - 1 JF - Nature Protocols TI - Immunocytochemical technique for protein localization in sections of plant tissues VL - 1 ER - TY - JOUR AB - Plant biology is currently confronted with an overflow of expression profile data provided by high-throughput microarray transcription analyses. However, the tissue and cellular resolution of these techniques is limited. Thus, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here we present an in situ mRNA hybridization method that is routinely used in the analysis of gene expression patterns. The protocol is optimized for mRNA localizations in sectioned tissue of Arabidopsis seedlings including embryos, roots, hypocotyls, young primary leaves and flowers. The detailed protocol, recommended controls and troubleshooting are presented along with examples of application. The total time for the process is 10 days. AU - Brewer, Philip B AU - Heisler, Marcus G AU - Hejátko, Jan AU - Jirí Friml AU - Eva Benková ID - 3014 IS - 3 JF - Nature Protocols TI - In situ hybridization for mRNA detection in Arabidopsis tissue sections VL - 1 ER - TY - JOUR AB - The basic concepts of the molecular machinery that mediates cell migration have been gleaned from cell culture systems. However, the three-dimensional environment within an organism presents migrating cells with a much greater challenge. They must move between and among other cells while interpreting multiple attractive and repulsive cues to choose their proper path. They must coordinate their cell adhesion with their surroundings and know when to start and stop moving. New insights into the control of these remaining mysteries have emerged from genetic dissection and live imaging of germ cell migration in Drosophila, zebrafish, and mouse embryos. In this review, we first describe germ cell migration in cellular and mechanistic detail in these different model systems. We then compare these systems to highlight the emerging principles. Finally, we contrast the migration of germ cells with that of immune and cancer cells to outline the conserved and different mechanisms. AU - Kunwar, Prabhat S AU - Daria Siekhaus AU - Lehmann, Ruth ID - 3152 JF - Annual Review of Cell and Developmental Biology TI - In vivo migration A germ cell perspective VL - 22 ER - TY - CONF AB - This paper presents an algorithm capable of real-time separation of foreground from background in monocular video sequences. Automatic segmentation of layers from colour/contrast or from motion alone is known to be error-prone. Here motion, colour and contrast cues are probabilistically fused together with spatial and temporal priors to infer layers accurately and efficiently. Central to our algorithm is the fact that pixel velocities are not needed, thus removing the need for optical flow estimation, with its tendency to error and computational expense. Instead, an efficient motion vs non-motion classifier is trained to operate directly and jointly on intensity-change and contrast. Its output is then fused with colour information. The prior on segmentation is represented by a second order, temporal, Hidden Markov Model, together with a spatial MRF favouring coherence except where contrast is high. Finally, accurate layer segmentation and explicit occlusion detection are efficiently achieved by binary graph cut. The segmentation accuracy of the proposed algorithm is quantitatively evaluated with respect to existing ground-truth data and found to be comparable to the accuracy of a state of the art stereo segmentation algorithm. Fore-ground/background segmentation is demonstrated in the application of live background substitution and shown to generate convincingly good quality composite video. AU - Criminisi, Antonio AU - Cross, Geoffrey AU - Blake, Andrew AU - Vladimir Kolmogorov ID - 3189 TI - Bilayer segmentation of live video VL - 1 ER - TY - JOUR AB - Algorithms for discrete energy minimization are of fundamental importance in computer vision. In this paper, we focus on the recent technique proposed by Wainwright et al. (Nov. 2005)- tree-reweighted max-product message passing (TRW). It was inspired by the problem of maximizing a lower bound on the energy. However, the algorithm is not guaranteed to increase this bound - it may actually go down. In addition, TRW does not always converge. We develop a modification of this algorithm which we call sequential tree-reweighted message passing. Its main property is that the bound is guaranteed not to decrease. We also give a weak tree agreement condition which characterizes local maxima of the bound with respect to TRW algorithms. We prove that our algorithm has a limit point that achieves weak tree agreement. Finally, we show that, our algorithm requires half as much memory as traditional message passing approaches. Experimental results demonstrate that on certain synthetic and real problems, our algorithm outperforms both the ordinary belief propagation and tree-reweighted algorithm in (M. J. Wainwright, et al., Nov. 2005). In addition, on stereo problems with Potts interactions, we obtain a lower energy than graph cuts. AU - Vladimir Kolmogorov ID - 3190 IS - 10 JF - IEEE Transactions on Pattern Analysis and Machine Intelligence TI - Convergent tree reweighted message passing for energy minimization VL - 28 ER - TY - CONF AB - We introduce the term cosegmentation which denotes the task of segmenting simultaneously the common parts of an image pair. A generative model for cosegmentation is presented. Inference in the model leads to minimizing an energy with an MRF term encoding spatial coherency and a global constraint which attempts to match the appearance histograms of the common parts. This energy has not been proposed previously and its optimization is challenging and NP-hard. For this problem a novel optimization scheme which we call trust region graph cuts is presented. We demonstrate that this framework has the potential to improve a wide range of research: Object driven image retrieval, video tracking and segmentation, and interactive image editing. The power of the framework lies in its generality, the common part can be a rigid/non-rigid object (or scene), observed from different viewpoints or even similar objects of the same class. AU - Rother, Carsten AU - Vladimir Kolmogorov AU - Minka, Thomas P AU - Blake, Andrew ID - 3188 TI - Cosegmentation of image pairs by histogram matching - Incorporating a global constraint into MRFs ER - TY - CONF AB - The Feistel-network is a popular structure underlying many block-ciphers where the cipher is constructed from many simpler rounds, each defined by some function which is derived from the secret key. Luby and Rackoff showed that the three-round Feistel-network – each round instantiated with a pseudorandom function secure against adaptive chosen plaintext attacks (CPA) – is a CPA secure pseudorandom permutation, thus giving some confidence in the soundness of using a Feistel-network to design block-ciphers. But the round functions used in actual block-ciphers are – for efficiency reasons – far from being pseudorandom. We investigate the security of the Feistel-network against CPA distinguishers when the only security guarantee we have for the round functions is that they are secure against non-adaptive chosen plaintext attacks (nCPA). We show that in the information-theoretic setting, four rounds with nCPA secure round functions are sufficient (and necessary) to get a CPA secure permutation. Unfortunately, this result does not translate into the more interesting pseudorandom setting. In fact, under the so-called Inverse Decisional Diffie-Hellman assumption the Feistel-network with four rounds, each instantiated with a nCPA secure pseudorandom function, is in general not a CPA secure pseudorandom permutation. AU - Maurer, Ueli M AU - Oswald, Yvonne A AU - Krzysztof Pietrzak AU - Sjödin, Johan ID - 3214 TI - Luby Rackoff ciphers from weak round functions VL - 4004 ER -