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

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

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

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk

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

Finding order in chaos : Dissecting single-cell heterogeneity in space and time

The cell is the smallest unit of life and contains DNA, RNA, proteins and a variety of other macromolecules. In recent years, technological advances in the field of single cell biology have revealed a staggering amount of phenotypic heterogeneity between cells in a population, which were previously considered homogenous. Previous work has largely been focused on studies of RNA. As proteins however are the ultimate effectors of genetic information, this thesis aims to provide a protein-centered view on cellular heterogeneity, particularly focusing on cell cycle and cellular metabolism.Most of my work has been performed within the framework of the Human Protein Atlas project. In the context of this project, we mapped the spatial distribution of more than 13.000 human proteins with subcellular resolution and found that around a quarter of all human proteins exhibit protein expression heterogeneity.In Paper I, we hypothesized that a majority of the observed cellular heterogeneity can be explained by differences in cell cycle progression. Therefore, we generated a map of proteomic and transcriptomic heterogeneity at subcellular resolution, which we precisely aligned to the cell cycle position of individual cells. This approach allowed us to identify hundreds of previously unknown cell cycle-related proteins. With sustained proliferative signaling representing a hallmark of cancer, novel cell-cycle proteins could serve as potential new drug targets against cancer. We further show that a large part of cell cycle dependent proteome variability is not established by transcriptomic cycling. This suggests that post-translational modifications are a major contributor to the regulation of cell cycle dependent protein level changes. Therefore, in Paper II, we carried out a deep phosphoproteome mass spectrometry profiling of the same cellular model as in Paper I and identified almost 5,000 cell cycle dependent phosphosites on over 2,000 proteins. The unprecedented scale of our phosphoproteomic data allows us to link cell cycle dependent protein expression dynamics to phosphorylation events. Furthermore, we identify a large set of proteins with stable expression levels and fluctuating phosphorylation patterns along cell cycle progression that likely alters protein function.Despite identifying hundreds of novel cell cycle dependent proteins in paper I, we observed that the majority of heterogeneously expressed proteins display variable expression independent of cell cycle progression, among them a large number of metabolic enzymes. Thus, we sought to describe the extent of subcellular metabolic complexity in human cells and tissues in Paper III. While we confirm metabolic compartmentalization in our dataset, we show that around 50% of metabolic enzymes localize to multiple cellular compartments. By integrating public protein-protein interaction data with our subcellular location information, we identify several enzymes with novel compartment-specific functions. Additionally, we observe a strongly elevated number of heterogeneously expressed enzymes compared to the background of the human proteome that is largely independent of cell cycle progression. We show that this heterogeneity can be manifested in the lineage of a single cell and is conserved in situ. To conclude, we suggest that the extensive metabolic heterogeneity can establish functional metabolic states in a population of human cells.Finally, in Paper IV, we assessed the heterogeneity of the mitochondrial proteome as they are metabolic powerhouses containing an elevated number of cell cycle independent variably expressed proteins. In this study, we correlated the variable expression of over 400 mitochondrial proteins to the expression of rate limiting enzymes in important mitochondrial pathways; such as the TCA cycle and ROS metabolism. We show that enzymes in the same pathways often correlate in their expression, indicating that their expression variability may contribute to the establishment of metabolic states.Altogether, the thesis illuminates the spatiotemporal complexity of the human proteome established by protein multilocalization and expression heterogeneity as fundamental non-genetic means of functional cell regulation.Cellen är den minsta enheten av liv, och varje cell innehåller en komplex uppsättning av DNA, RNA, proteiner och andra makromolekyler. Under de senaste åren har teknologiska framsteg inom cellbiologiska studier av enskilda celler avslöjat en överväldigande mängd fenotypisk heterogenitet mellan cellpopulationer som tidigare betraktades som homogena. Tidigare kartläggning av denna heterogenitet har främst fokuserat på studier av RNA. Denna avhandling syftar dock till att ge en proteincentrerad syn på cellulär variabilitet, med särskilt fokus på cellcykeln och cellulär metabolism.Det mesta av mitt arbete innefattar data från Human Protein Atlas-projektet. I detta projekt har vi kartlagt den spatiala fördelningen av över 13 000 mänskliga proteiner med subcellulär upplösning. Vi finner att ungefär en fjärdedel av alla mänskliga proteiner uppvisar heterogenitet i proteinuttryck mellan enskilda celler.I Artikel I undersökte vi hypotesen att majoriteten av den observerade cellulära heterogeniteten kan förklaras av skillnader i cellcykelprogression. Därför genererade vi en stor karta över proteomisk och transkriptomisk uttrycksdata i relation till den mänskliga cellcykeln i enskilda celler. Vi identifierade hundratals nya proteiner relaterade till cellcykeln, vilka kan komma att fungera som potentiella läkemedelsmål vid sjukdomar som cancer. Vi visar vidare att en stor del av den cellcykelberoende variabiliteten på proteinnivå inte etableras genom cykliska ändringar av transkriptomet. Detta antyder att posttranslationella modifieringar i betydande utsträckning bidrar till regleringen av dynamiska förändringar i proteinuttryck under cellcykeln.Därför utförde vi i Artikel II en djup masspektrometriprofilering av fosfoproteomet längs samma cellulära modell som i Artikel I och identifierade över 2000 cellcykelberoende fosforyleringsplatser hos människans proteiner. Den oöverträffade omfattningen av denna fosfoproteomiska data gör att vi kan koppla cellcykelberoende dynamik i proteinuttryck till fosforyleringshändelser. Dessutom identifierar vi en stor uppsättning proteiner med stabila uttrycksnivåer och varierande fosforyleringsmönster längs cellcykelns progression, vilket sannolikt reglerar deras funktion.I Artikel I observerade vi också att majoriteten av heterogent uttryckta proteiner visar variation som är oberoende av cellcykelprogression, och bland dessa proteiner finns ett stort antal metaboliska enzymer. Därför strävade vi efter att beskriva omfattningen av subcellulär metabolisk komplexitet hos mänskliga celler och vävnader i Artikel III. Samtidigt som vi bekräftar att det finns en spatiell uppdelning av cellulära metaboliska processer visar vi att ungefär 50% av de metaboliska enzymerna lokaliserar till flera subcellulära avdelningar. Genom att integrera offentliga protein-protein-interaktionsdata med vår information om subcellulär lokalisering identifierar vi flera enzymer som utför olika funktioner i olika cellulära avdelningar. Dessutom observerar vi en starkt ökad mängd heterogent uttryckta enzymer. Vi föreslår att denna omfattande metaboliska heterogenitet kan etablera olika funktionella metabola tillstånd i en population av mänskliga celler.Slutligen utvärderade vi i Artikel IV heterogeniteten hos det mitokondriella proteomet eftersom de är metabola kraftverk som innehåller en stor andel proteiner vars uttryck varierar beroende av cellcykeln. I denna studie korrelerade vi det variabla uttrycket av över 400 mitokondriella proteiner med uttrycket av hastighetsbegränsande enzymer i viktiga mitokondriella processer, såsom citronsyracykeln och metabolism av reaktiva syreföreningar. Vi visar att enzymer i samma reaktionsvägar ofta korrelerar i sitt uttryck, vilket indikerar att deras uttrycksvariabilitet kan bidra till etableringen av metabola tillstånd.</p

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High conductance voltage-and Ca(2+)-activated K(+) channels (Slo1 or BK channels) function in many physiological processes that link cell membrane voltage and intracellular Ca(2+), including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning(1–8). Like other voltage-dependent K(+) (Kv) channels, BK channels open when the cell membrane depolarizes, but in contrast to other Kv channels they also open when intracellular Ca(2+) levels rise. Channel opening by Ca(2+) is conferred by a structure called the gating ring, located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed conformation of the gating ring, but the open conformation is not yet known(9). Here we present the Ca(2+)-bound, open conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The magnitude of opening explains how Ca(2+) binding can open the pore. These findings present amolecular basis of Ca(2+) activation and suggest new possibilities for targeting the gating ring to treat diseases such as asthma and hypertension

Self-Supervised Deep Learning Encodes High-Resolution Features of Protein Subcellular Localization

AbstractElucidating the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here, we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. cytoself leverages a self-supervised training scheme that does not require pre-existing knowledge, categories, or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes such as nuclear, cytoplasmic and vesicular, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoself’s ability to cluster proteins into organelles and protein complex clusters using a clustering score, and show that cytoself attains higher scores than previous unsupervised or self-supervised approaches. Finally, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret these features in the context of the fluorescence images, and analyze the performance contributions of the different components of our approach.</jats:p

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

AbstractA central challenge of the post-genomic 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/sgRNA 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. 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.SIGNIFICANCE STATEMENTThe function of a large fraction of the human proteome still remains poorly characterized. Tagging proteins with a functional sequence is a powerful way to access function, and inserting tags at endogenous genomic loci allows the preservation of a near-native cellular background. To characterize the cellular role of human proteins in a systematic manner and in a native context, we developed a method for tagging endogenous human proteins with GFP that is both rapid and readily applicable at a genome-wide scale. Our approach allows studying both localization and interaction partners of the protein target. Our results pave the way for the large-scale generation of endogenously tagged human cell lines for a systematic functional interrogation of the human proteome.</jats:sec