Protein Delivery into Plant Cells: Toward In vivo Structural Biology.

Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily controlled while dealing with an isolated, purified protein, a serious shortcoming of such in vitro work is that we cannot mimic the extremely complex intracellular environment in which the protein exists and functions. Therefore, it is highly desirable to investigate proteins in their natural habitat, i.e., within live cells. This is the major ambition of in-cell NMR, which aims to approach structure-function relationship under true in vivo conditions following delivery of labeled proteins into cells under physiological conditions. With a multidisciplinary approach that includes recombinant protein production, confocal fluorescence microscopy, nuclear magnetic resonance (NMR) spectroscopy and different intracellular protein delivery strategies, we explore the possibility to develop in-cell NMR studies in living plant cells. While we provide a comprehensive framework to set-up in-cell NMR, we identified the efficient intracellular introduction of isotope-labeled proteins as the major bottleneck. Based on experiments with the paradigmatic intrinsically disordered proteins (IDPs) Early Response to Dehydration protein 10 and 14, we also established the subcellular localization of ERD14 under abiotic stress.

1H, 15N, 13C resonance assignment of plant dehydrin early response to dehydration 10 (ERD10).

Early response to dehydration 10 protein (ERD10) is an intrinsically disordered protein from Arabidopsis thaliana. The protein is upregulated during stress however its mechanism of action at atomic level is not well understood. In the present work multidimensional NMR methodologies are used in order to facilitate the process of chemical shift assignment. The information provided here supports further NMR spectroscopy experiments aimed at elucidation of ERD10 behaviour during molecular recognition events with other proteins.

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression.

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases.

Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.

Detection and Analysis of SUMOylation Substrates In Vitro and In Vivo.

SUMOylation is a widely used protein posttranslational mechanism capable of regulating substrates localization, stability, and/or activity. Identification and characterization of bona fide SUMO substrates is a laborious task but its discovery can shed light to exquisite and crucial regulatory signaling events occurring within the cell. Experiments performed in the SUMOylation field often demand a good understanding of the putative substrate's function and necessitate a solid knowledge regarding both in vitro and in vivo approaches. This contribution offers a simplified view into some of the most common experiments performed in biochemical and cell biological research of the SUMO pathway in mammalian systems. It also summarizes and updates well established protocols and tricks in order to improve the likelihood to obtain reliable and reproducible results.

Towards Understanding Protein Disorder In-Cell.

Investigating the activity and structure of cellular biochemical machinery at atomic resolution has been a point of paramount significance for understanding health and disease over the decades. The underlying molecular mechanisms are primarily studied in vitro. Nuclear magnetic resonance (NMR) is a technique that allows to look into cells and study proteins and other constituents, thanks to careful experimental design and technological advances (spectrometer sensitivity and pulse sequence design). Here we outline current applications of the technique and propose a realistic future for the field.

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs.