Structural and Functional Studies of SLO K+ Channels: Mechanisms of Gating by Intracellular Signaling

Eukaryotic K+ channels from the SLO family (SLO1, SLO2 and SLO3) provide a link between intracellular signaling and the electrical activity of a cell. The opening and closing (gating) of the three different SLO homologs is controlled by the synergistic action of membrane voltage and specific intracellular cues: Ca2+ binding in SLO1, Na+ binding in SLO2 and pH increase in SLO3. It is known that intracellular signals activate SLO channels by acting on the large cytoplasmic domains (CTDs) of these proteins, which follows the transmembrane ionconduction pore. However, a molecular description of the mechanisms of intracellular gating in SLO channels is still lacking. In this thesis, I present biochemical, structural and functional studies aiming at understanding how the activity of SLO1 and SLO3 channels is controlled by intracellular Ca2+ binding and pH increase, respectively. First, I describe recombinant methods for the large-scale expression and purification of functional SLO channels, paving the way for a more complete biochemical and structural analysis of these proteins. Then, I report the crystal structures of the large cytoplasmic domains (CTDs) from two different SLO1 channels. Structures of the Ca2+-bound CTDs from human and zebrafish SLO1 channels define the precise molecular architecture of SLO1’s Ca2+-sensing module: CTDs from the four subunits of a tetrameric SLO1 channel assemble in a so-called gating ring structure at the intracellular face of the membrane. In conjunction with other studies, these results describe how Ca2+ binding affects the conformation of one layer of the SLO1 gating ring, which can explain the Ca2+-driven opening of SLO1’s ion conduction pore. Next, I present the crystal structure of the human SLO3 gating ring. A comparison with the SLO1 structures suggests that the hSLO3 structure represents the open conformation of the hSLO3 gating ring. Finally, I describe functional mutagenesis studies on the mouse SLO3 ortholog, which reveal a possible mechanism for pH sensing in the mouse SLO3 channel. Surprisingly, the mechanism I propose appears not to be conserved in SLO3 channels from other species. This could be a dramatic example of how new functional mechanisms can easily evolve within the very versatile scaffold of a gating ring structure. Altogether, the results presented in this thesis provide a molecular framework to understand the mechanisms of intracellular gating in SLO channels

Improved split fluorescent proteins for endogenous protein labeling.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use

Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

La renovación de la palabra en el bicentenario de la Argentina : los colores de la mirada lingüística

El libro reúne trabajos en los que se exponen resultados de investigaciones presentadas por investigadores de Argentina, Chile, Brasil, España, Italia y Alemania en el XII Congreso de la Sociedad Argentina de Lingüística (SAL), Bicentenario: la renovación de la palabra, realizado en Mendoza, Argentina, entre el 6 y el 9 de abril de 2010. Las temáticas abordadas en los 167 capítulos muestran las grandes líneas de investigación que se desarrollan fundamentalmente en nuestro país, pero también en los otros países mencionados arriba, y señalan además las áreas que recién se inician, con poca tradición en nuestro país y que deberían fomentarse. Los trabajos aquí publicados se enmarcan dentro de las siguientes disciplinas y/o campos de investigación: Fonología, Sintaxis, Semántica y Pragmática, Lingüística Cognitiva, Análisis del Discurso, Psicolingüística, Adquisición de la Lengua, Sociolingüística y Dialectología, Didáctica de la lengua, Lingüística Aplicada, Lingüística Computacional, Historia de la Lengua y la Lingüística, Lenguas Aborígenes, Filosofía del Lenguaje, Lexicología y Terminología

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context

Improved split fluorescent proteins for endogenous protein labeling.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use

A scalable strategy for high-throughput GFP tagging of endogenous human proteins

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

<p>Zenodo hosts the archival version of this document; for convenient viewing, please visit <a href="http://andrewgyork.github.io/split_wrmscarlet" target="_blank" rel="noopener">andrewgyork.github.io/split_wrmscarlet </a>or <a href="http://marimar128.github.io/split_wrmscarlet" target="_blank" rel="noopener">marimar128.github.io/split_wrmscarlet</a>.</p> <h4><a href="https://andrewgyork.github.io/split_wrmscarlet/#Abstract" target="_self">Abstract</a></h4> <p>We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous <em>C. elegans</em> proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of <em>C. elegans</em> proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically-split fluorescent proteins solve this problem in <em>C. elegans</em>: the small fragment can quickly and easily be fused to almost any protein of interest and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available <em>C. elegans</em> lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, <a href="https://www.addgene.org/138966/">split-wrmScarlet</a>. We generate transgenic <em>C. elegans</em> lines to allow easy <a href="https://cgc.umn.edu/strain/CF4582">single-color</a> labeling in <a href="https://cgc.umn.edu/strain/CF4610">muscle</a> or <a href="https://cgc.umn.edu/strain/DUP237">germline</a> and <a href="https://cgc.umn.edu/strain/CF4588">dual-color</a> labeling in somatic cells. We also describe 'glonads', a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a <a href="https://doi.org/10.17504/protocols.io.bamkic4w">protocol</a> for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at <a href="https://doi.org/10.5281/zenodo.3993663">doi.org/10.5281/zenodo.3993663</a></p&gt