Development of Bimolecular Fluorescence Complementation reagents for the detection of Arabidopsis thaliana KAT1 protein-protein interactions using the Golden Braid cloning system

[EN] KAT1 is an Arabidopsis thaliana potassium voltage-gated channel of the Shaker family. This ion channel is fundamental for the control of membrane conductance in guard cells, leading to stomatal opening or closing in response to environmental changes. The stomatal movement controls the gas exchange, as well as the amount of water lost due to transpiration. Therefore, the underlying mechanisms of these stomatal movements will likely be influenced by proteins that regulate KAT1 activity. The Shaker channels have been proposed to form functional channels associating into heterotetramers or homotetramers, but our knowledge regarding regulators of these potassium channels is very limited. From a previous yeast-based high-throughput screening performed in our laboratory, a list of 14 possible KAT1 interacting proteins was obtained. To determine the physiological relevance of the identified interacting proteins, these interactions need to be confirmed in plant cells. The Bimolecular Fluorescence Complementation (BiFC) technique is based on YFP’s ability to recover its fluorescence upon physical approximation of its two halves. Using this technique, both the validity of the KAT1 interacting proteins and also the subcellular location of these interactions in the plant can be analyzed by confocal microscopy. GoldenBraid is a cloning system that allows the modular construction of multigenic DNA structures, and it provides a fast and efficient platform to assemble all the required pieces for the BiFC analysis and to insert them into a single multigenic construction. GoldenBraid is flexible enough to allow the construction of all desired fusion protein combinations. A well-established BiFC cloning procedure will be fundamental to study the protein-protein interactions of the candidate proteins because of the large number of possible combinations of fusion proteins required for these analyses (8 combinations for each interaction). On the one hand, this project developed and implemented the KAT1 sequence as a GoldenBraid part and it was successfully tested in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated transformation followed by confocal microscopy. And on the other hand, non-functional combinations for the BiFC assay were studied analyzing the expression and localization of KAT1-YFP and YFP-KAT1 fusion proteins which led to discard N-terminal fusions with KAT1.[CA] KAT1 és un canal de potassi regulat per voltatge d’Arabidopsis thaliana que pertany a la família Shaker. Aquest canal de ions és fonamental per al control de la conductància a la membrana de les cèl·lules guarda, causant l’obertura o el tancament dels estomes en resposta als canvis ambientals. El moviment dels estomes controla l’intercanvi de gasos, així com la perduda d’aigua per transpiració. Per aquesta raó, els mecanismes subjacents als moviments estomàtics estan probablement influenciats per les proteïnes que regulen l’activitat de KAT1. S’ha proposat que els canals Shaker s’associen formant heterotetramers o homotetramers per tal de formar un canal funcional, però el nostre coneixement sobre els reguladors d’aquests canals de potassi és molt limitat. A partir d’un cribratge d’alt rendiment basat en llevat realitzat al nostre laboratori, es va obtindré una llista de 14 possible proteïnes que interaccionen amb KAT1. Per tal de determinar la rellevància fisiològica de les possibles proteïnes que interaccionen amb KAT1, aquestes interaccions han de ser confirmades en cèl·lules vegetals. La Complementació Bimolecular de Fluorescència (BiFC) és una tècnica basada en l’habilitat de YFP, que és capaç de recobrar la seva fluorescència per la aproximació de les seves dues meitats. Utilitzant aquesta tècnica, la validesa de les proteïnes que interaccionen amb KAT1 i també la localització subcel·lular d’aquestes interaccions en la planta podrà ser analitzada mitjançant microscòpia confocal. GoldenBraid es un sistema de clonatge que permet la construcció de manera modular d’estructures de DNA, i proporciona una plataforma ràpida i eficient per muntar totes les peces que es requereixen per a realitzar la BiFC i insertar-les en una sola unitat multigènica. El sistema GoldenBraid és suficientment flexible com per a permetre la construcció de totes les combinacions de proteïnes de fusió. Un protocol ben establert de BiFC serà fonamental per estudiar les interaccions proteïna-proteïna de les proteïnes candidates degut a l’elevat nombre de combinacions possibles de proteïnes de fusió que es requereixen per a aquests anàlisis (8 combinacions per a cada interacció). Per un costat, aquest projecte ha desenvolupat i implementat la seqüència de KAT1 com a una part del GoldenBraid i va ser provada amb èxit en Nicotiana benthamiana utilitzant una transformació mitjançant Agrobacterium tumefaciens seguida del anàlisis per microscòpia confocal. I per altre costat, les combinacions no funcionals per a l’assaig de BiFC sigueren estudiades analitzant l’expressió i localització de les proteïnes de fusió KAT1-YFP i YFP-KAT1, fets que portaren a descartar les fusions N-terminals de KAT1.Mossi Albiach, A. (2016). Development of Bimolecular Fluorescence Complementation reagents for the detection of Arabidopsis thaliana KAT1 protein-protein interactions using the Golden Braid cloning system. http://hdl.handle.net/10251/67656.TFG

Imaging the spatial organization of healthy human brain and glioblastoma

Multicellular biological systems have a single genetic plan that contains the set of instructions to shape the three-dimensional architecture of specialized tissues and organs. The genetic plan is contained in the DNA, and information is relayed through RNA to produce proteins that grant cells with the power to carry out different functions. As cells specialize giving rise to differentiated cell types of a particular tissue, they express different RNA molecules that will reshape their protein content and grant them with a certain phenotype. The development of scRNA-seq technologies at the start of the 2010s allowed researcher to quantify RNA expression at high gene throughput, enabling a deep molecular characterization of cell types. However, scRNA-seq methods requires dissociation of tissues and organs into cell suspensions, and the spatial organization is lost. The present thesis features the need for high-throughput spatial transcriptomics methods, to enable the characterization of cell types and the spatial architecture of the adult human healthy brain and adult diffuse high-grade gliomas. The thesis is composed of three parts: first, the evolution of the spatial transcriptomics methods and field; second, cell types of the human healthy brain; and third, the biology of adult high- grade diffuse gliomas, and more specifically glioblastoma. In paper I we present Enhanced Electric Fluorescent In Situ Hybridization, or EEL- FISH, a high-throughput spatial transcriptomics methods that enables to obtain the expression profile of up to 448 RNA species per fluorescent channel over large tissue areas. The ability speedily to image over large tissue area was achieved by enhancing the RNA capture on a surface, which reduced unnecessary imaging time through the tissue thickness (z-axis). Moreover, EEL-FISH enabled imaging of human tissue sections, which are typically challenging for smFISH due to their high autofluorescence. EEL FISH reduces tissue autofluorescence because the tissue is removed after RNA has been captured. Paper II focuses on the direct application of single cell technologies to characterize the cell types of over 100 regions of the human adult brain of healthy individuals. To this end, we employed scRNA-seq to create a census of cell types and combined with EEL-FISH to characterize the spatial organization of cells in specific brain areas. Lastly, we used EEL-FISH to understand the spatial organization of adult high- grade gliomas, more specifically glioblastoma, in paper III. We characterized the expression of 888 RNA species, by using two fluorescent channels, of 57 sections from 27 different high-grade glioma patients (2 oligodendroglioma and 25 glioblastoma). We generalized a model of the spatial organization of glioblastoma, which organizes across a gradient of hypoxia and wound response that are activated on top of a glial-like tumour cell. Along this thesis, I will review the literature of spatial transcriptomics, and the biology of the adult human brain and glioblastoma from a single cell perspective, and discuss the results of the research conducted in papers I, II and III with respect to these topics

Molecular architecture of the developing mouse brain.

The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1-8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system