Publications by authors named "Prisca Liberali"

25 Publications

  • Page 1 of 1

Cell fate coordinates mechano-osmotic forces in intestinal crypt formation.

Nat Cell Biol 2021 Jul 21;23(7):733-744. Epub 2021 Jun 21.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

Intestinal organoids derived from single cells undergo complex crypt-villus patterning and morphogenesis. However, the nature and coordination of the underlying forces remains poorly characterized. Here, using light-sheet microscopy and large-scale imaging quantification, we demonstrate that crypt formation coincides with a stark reduction in lumen volume. We develop a 3D biophysical model to computationally screen different mechanical scenarios of crypt morphogenesis. Combining this with live-imaging data and multiple mechanical perturbations, we show that actomyosin-driven crypt apical contraction and villus basal tension work synergistically with lumen volume reduction to drive crypt morphogenesis, and demonstrate the existence of a critical point in differential tensions above which crypt morphology becomes robust to volume changes. Finally, we identified a sodium/glucose cotransporter that is specific to differentiated enterocytes that modulates lumen volume reduction through cell swelling in the villus region. Together, our study uncovers the cellular basis of how cell fate modulates osmotic and actomyosin forces to coordinate robust morphogenesis.
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http://dx.doi.org/10.1038/s41556-021-00700-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611267PMC
July 2021

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

Cell Stem Cell 2021 Jun 11. Epub 2021 Jun 11.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.
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http://dx.doi.org/10.1016/j.stem.2021.05.013DOI Listing
June 2021

Phenotypic landscape of intestinal organoid regeneration.

Nature 2020 10 7;586(7828):275-280. Epub 2020 Oct 7.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.
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http://dx.doi.org/10.1038/s41586-020-2776-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116869PMC
October 2020

Design principles of tissue organisation: How single cells coordinate across scales.

Curr Opin Cell Biol 2020 12 2;67:37-45. Epub 2020 Sep 2.

Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058, Basel, Switzerland; University of Basel, Petersplatz 1, 4001, Basel, Switzerland. Electronic address:

Cells act as building blocks of multicellular organisms, forming higher-order structures at different biological scales. Niches, tissues and, ultimately, entire organisms consist of single cells that remain in constant communication. Emergence of developmental patterns and tissue architecture thus relies on single cells acting as a collective, coordinating growth, migration, cell fate transitions and cell type sorting. For this, information has to be transmitted forward from cells to tissues and fed back to the individual cell to allow dynamic and robust coordination. Here, we define the design principles of tissue organisation integrating chemical, genetic and mechanical cues. We also review the state-of-the-art technologies used for dissecting collective cellular behaviours at single cell- and tissue-level resolution. We finally outline future challenges that lie in a comprehensive understanding of how single cells coordinate across biological scales to insure robust development, homoeostasis and regeneration of tissues.
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http://dx.doi.org/10.1016/j.ceb.2020.07.004DOI Listing
December 2020

Engineering human knock-in organoids.

Nat Cell Biol 2020 03;22(3):261-263

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

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http://dx.doi.org/10.1038/s41556-020-0478-zDOI Listing
March 2020

Exploring single cells in space and time during tissue development, homeostasis and regeneration.

Development 2019 06 27;146(12). Epub 2019 Jun 27.

Department of Quantitative Biology, Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, 4058 Basel, Switzerland

Complex 3D tissues arise during development following tightly organized events in space and time. In particular, gene regulatory networks and local interactions between single cells lead to emergent properties at the tissue and organism levels. To understand the design principles of tissue organization, we need to characterize individual cells at given times, but we also need to consider the collective behavior of multiple cells across different spatial and temporal scales. In recent years, powerful single cell methods have been developed to characterize cells in tissues and to address the challenging questions of how different tissues are formed throughout development, maintained in homeostasis, and repaired after injury and disease. These approaches have led to a massive increase in data pertaining to both mRNA and protein abundances in single cells. As we review here, these new technologies, in combination with live imaging, now allow us to bridge spatial and temporal information quantitatively at the single cell level and generate a mechanistic understanding of tissue development.
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http://dx.doi.org/10.1242/dev.176727DOI Listing
June 2019

Self-organization and symmetry breaking in intestinal organoid development.

Nature 2019 05 24;569(7754):66-72. Epub 2019 Apr 24.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.
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http://dx.doi.org/10.1038/s41586-019-1146-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544541PMC
May 2019

Primed Track, high-fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins.

Elife 2019 01 21;8. Epub 2019 Jan 21.

Department for Biosystems Science and Engineering (D-BSSE), ETH Zurich, Basel, Switzerland.

Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased imaging time intervals - addressing major concerns in the field of volumetric embryo imaging.
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http://dx.doi.org/10.7554/eLife.44491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340703PMC
January 2019

From single cells to tissue self-organization.

FEBS J 2019 04 19;286(8):1495-1513. Epub 2018 Nov 19.

Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland.

Self-organization is a process by which interacting cells organize and arrange themselves in higher order structures and patterns. To achieve this, cells must have molecular mechanisms to sense their complex local environment and interpret it to respond accordingly. A combination of cell-intrinsic and cell-extrinsic cues are decoded by the single cells dictating their behaviour, their differentiation and symmetry-breaking potential driving development, tissue remodeling and regenerative processes. A unifying property of these self-organized pattern-forming systems is the importance of fluctuations, cell-to-cell variability, or noise. Cell-to-cell variability is an inherent and emergent property of populations of cells that maximize the population performance instead of the individual cell, providing tissues the flexibility to develop and maintain homeostasis in diverse environments. In this review, we will explore the role of self-organization and cell-to-cell variability as fundamental properties of multicellularity-and the requisite of single-cell resolution for its understanding. Moreover, we will analyze how single cells generate emergent multicellular dynamics observed at the tissue level 'travelling' across different scales: spatial, temporal and functional.
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http://dx.doi.org/10.1111/febs.14694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519261PMC
April 2019

Modifiers of prion protein biogenesis and recycling identified by a highly parallel endocytosis kinetics assay.

J Biol Chem 2017 05 24;292(20):8356-8368. Epub 2017 Mar 24.

Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland. Electronic address:

The cellular prion protein, PrP, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrP is the causative agent of prion diseases. Conversion of PrP into PrP is thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrP may, therefore, help elucidate the conversion process. Here we describe a time-resolved fluorescence energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrP The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrP trafficking. Simultaneous administration of FRET donor and acceptor anti-PrP antibodies to living cells yielded a measure of PrP surface density, whereas sequential addition of each antibody visualized the internalization rate of PrP (Z' factor >0.5). RNA interference assays showed that suppression of AP2M1 (AP-2 adaptor protein), RAB5A, VPS35 (vacuolar protein sorting 35 homolog), and M6PR (mannose 6-phosphate receptor) blocked PrP internalization, whereas down-regulation of GIT2 and VPS28 increased PrP internalization. PrP cell-surface expression was reduced by down-regulation of RAB5A, VPS28, and VPS35 and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrP trafficking and demonstrate the power of this assay for identifying modulators of PrP trafficking.
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http://dx.doi.org/10.1074/jbc.M116.773283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437241PMC
May 2017

Trajectories of cell-cycle progression from fixed cell populations.

Nat Methods 2015 Oct 24;12(10):951-4. Epub 2015 Aug 24.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

An accurate dissection of sources of cell-to-cell variability is crucial for quantitative biology at the single-cell level but has been challenging for the cell cycle. We present Cycler, a robust method that constructs a continuous trajectory of cell-cycle progression from images of fixed cells. Cycler handles heterogeneous microenvironments and does not require perturbations or genetic markers, making it generally applicable to quantifying multiple sources of cell-to-cell variability in mammalian cells.
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http://dx.doi.org/10.1038/nmeth.3545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004611PMC
October 2015

Sumoylation regulates EXO1 stability and processing of DNA damage.

Cell Cycle 2015 Aug 17;14(15):2439-50. Epub 2015 Jun 17.

a Institute of Molecular Cancer Research; University of Zurich ; Zurich , Switzerland.

DNA double-strand break repair by the error-free pathway of homologous recombination (HR) requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM are responsible for the extensive resection of DNA ends to produce 3'-overhangs, which are essential intermediates for downstream steps of HR. Here we show that EXO1 is a SUMO target and that sumoylation affects EXO1 ubiquitylation and protein stability. We identify an UBC9-PIAS1/PIAS4-dependent mechanism controlling human EXO1 sumoylation in vivo and demonstrate conservation of this mechanism in yeast by the Ubc9-Siz1/Siz2 using an in vitro reconstituted system. Furthermore, we show physical interaction between EXO1 and the de-sumoylating enzyme SENP6 both in vitro and in vivo, promoting EXO1 stability. Finally, we identify the major sites of sumoylation in EXO1 and show that ectopic expression of a sumoylation-deficient form of EXO1 rescues the DNA damage-induced chromosomal aberrations observed upon wt-EXO1 expression. Thus, our study identifies a novel layer of regulation of EXO1, making the pathways that regulate its function an ideal target for therapeutic intervention.
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http://dx.doi.org/10.1080/15384101.2015.1060381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615030PMC
August 2015

Single-cell and multivariate approaches in genetic perturbation screens.

Nat Rev Genet 2015 Jan 2;16(1):18-32. Epub 2014 Dec 2.

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Large-scale genetic perturbation screens are a classical approach in biology and have been crucial for many discoveries. New technologies can now provide unbiased quantification of multiple molecular and phenotypic changes across tens of thousands of individual cells from large numbers of perturbed cell populations simultaneously. In this Review, we describe how these developments have enabled the discovery of new principles of intracellular and intercellular organization, novel interpretations of genetic perturbation effects and the inference of novel functional genetic interactions. These advances now allow more accurate and comprehensive analyses of gene function in cells using genetic perturbation screens.
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http://dx.doi.org/10.1038/nrg3768DOI Listing
January 2015

A hierarchical map of regulatory genetic interactions in membrane trafficking.

Cell 2014 Jun;157(6):1473-1487

Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Electronic address:

Endocytosis is critical for cellular physiology and thus is highly regulated. To identify regulatory interactions controlling the endocytic membrane system, we conducted 13 RNAi screens on multiple endocytic activities and their downstream organelles. Combined with image analysis of thousands of single cells per perturbation and their cell-to-cell variability, this created a high-quality and cross-comparable quantitative data set. Unbiased analysis revealed emergent properties of the endocytic membrane system and how its complexity evolved and distinct programs of regulatory control that coregulate specific subsets of endocytic uptake routes and organelle abundances. We show that these subset effects allow the mapping of functional regulatory interactions and their interaction motifs between kinases, membrane-trafficking machinery, and the cytoskeleton at a large scale, some of which we further characterize. Our work presents a powerful approach to identify regulatory interactions in complex cellular systems from parallel single-gene or double-gene perturbation screens in human cells and yeast.
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http://dx.doi.org/10.1016/j.cell.2014.04.029DOI Listing
June 2014

Predicting functional gene interactions with the hierarchical interaction score.

Nat Methods 2013 Nov 6;10(11):1089-92. Epub 2013 Oct 6.

1] Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland. [2].

Systems biology aims to unravel the vast network of functional interactions that govern biological systems. To date, the inference of gene interactions from large-scale 'omics data is typically achieved using correlations. We present the hierarchical interaction score (HIS) and show that the HIS outperforms commonly used methods in the inference of functional interactions between genes measured in large-scale experiments, making it a valuable statistic for systems biology.
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http://dx.doi.org/10.1038/nmeth.2655DOI Listing
November 2013

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS.

Proc Natl Acad Sci U S A 2013 Jun 28;110(24):9794-9. Epub 2013 May 28.

Institute of Protein Biochemistry, National Research Council, 80131 Naples, Italy.

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.
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http://dx.doi.org/10.1073/pnas.1222413110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683763PMC
June 2013

Towards quantitative cell biology.

Nat Cell Biol 2012 Dec;14(12):1233

Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Cell biologists must decide whether to embrace the maturing field of systems biology. We argue that a fusion of the two is urgently needed to strengthen both fields.
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http://dx.doi.org/10.1038/ncb2648DOI Listing
December 2012

Single-cell analysis of population context advances RNAi screening at multiple levels.

Mol Syst Biol 2012 Apr 24;8:579. Epub 2012 Apr 24.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.
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http://dx.doi.org/10.1038/msb.2012.9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361004PMC
April 2012

Population context determines cell-to-cell variability in endocytosis and virus infection.

Nature 2009 Sep 26;461(7263):520-3. Epub 2009 Aug 26.

Institute of Molecular Systems Biology, ETH Zurich (Swiss Federal Institute of Technology), Wolfgang Pauli-Strasse 16, CH-8093 Zurich, Switzerland.

Single-cell heterogeneity in cell populations arises from a combination of intrinsic and extrinsic factors. This heterogeneity has been measured for gene transcription, phosphorylation, cell morphology and drug perturbations, and used to explain various aspects of cellular physiology. In all cases, however, the causes of heterogeneity were not studied. Here we analyse, for the first time, the heterogeneous patterns of related cellular activities, namely virus infection, endocytosis and membrane lipid composition in adherent human cells. We reveal correlations with specific cellular states that are defined by the population context of a cell, and we derive probabilistic models that can explain and predict most cellular heterogeneity of these activities, solely on the basis of each cell's population context. We find that accounting for population-determined heterogeneity is essential for interpreting differences between the activity levels of cell populations. Finally, we reveal that synergy between two molecular components, focal adhesion kinase and the sphingolipid GM1, enhances the population-determined pattern of simian virus 40 (SV40) infection. Our findings provide an explanation for the origin of heterogeneity patterns of cellular activities in adherent cell populations.
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http://dx.doi.org/10.1038/nature08282DOI Listing
September 2009

Protein kinases: starting a molecular systems view of endocytosis.

Annu Rev Cell Dev Biol 2008 ;24:501-23

Institute of Molecular Systems Biology, ETH Zurich, CH-8093 Zurich, Switzerland.

The field of endocytosis is in strong need of formal biophysical modeling and mathematical analysis. At the same time, endocytosis must be much better integrated into cellular physiology to understand the former's complex behavior in such a wide range of phenotypic variations. Furthermore, the concept that endocytosis provides the space-time for signal transduction can now be experimentally addressed. In this review, we discuss these principles and argue for a systematic and top-down approach to study the endocytic membrane system. We provide a summary of published observations on protein kinases regulating endocytic machinery components and discuss global unbiased approaches to further map out kinase regulatory networks. In particular, protein phosphorylation is at the heart of controlling the physical properties of endocytosis and of integrating these physical properties into the signal transduction networks of the cell to allow a fine-tuned response to the continuously varying physiological conditions of a cell.
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http://dx.doi.org/10.1146/annurev.cellbio.041008.145637DOI Listing
December 2008

A Raft-derived, Pak1-regulated entry participates in alpha2beta1 integrin-dependent sorting to caveosomes.

Mol Biol Cell 2008 Jul 30;19(7):2857-69. Epub 2008 Apr 30.

Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, FI-40351 Jyväskylä, Finland.

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.
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http://dx.doi.org/10.1091/mbc.e07-10-1094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441652PMC
July 2008

The closure of Pak1-dependent macropinosomes requires the phosphorylation of CtBP1/BARS.

EMBO J 2008 Apr 20;27(7):970-81. Epub 2008 Mar 20.

Laboratory of Cell Regulation, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy.

Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS-induced fission to human viral infection.
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http://dx.doi.org/10.1038/emboj.2008.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323256PMC
April 2008

Steady-state and laser flash photolysis study of the carbon-carbon bond fragmentation reactions of 2-arylsulfanyl alcohol radical cations.

J Org Chem 2004 Nov;69(24):8323-30

Dipartimento di Chimica, Università La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.

The N-methylquinolinium tetrafluoroborate (NMQ(+))-sensitized photolysis of the erythro-1,2-diphenyl-2-arylsulfanylethanols 1-3 (1, aryl = phenyl; 2, aryl = 4-methylphenyl; 3, aryl = 3-chlorophenyl) has been investigated in MeCN, under laser flash and steady-state photolysis. Under laser irradiation, the formation of sulfide radical cations of 1-3, in the monomeric (lambda(max) = 520-540 nm) and dimeric form (lambda(max) = 720-->800 nm), was observed within the laser pulse. The radical cations decayed by first-order kinetics, and under nitrogen, the formation of ArSCH(*)Ph (lambda(max) = 350-360 nm) was clearly observed. This indicates that the decay of the radical cation is due to a fragmentation process involving the heterolytic C-C bond cleavage, a conclusion fully confirmed by steady-state photolysis experiments (formation of benzaldehyde and the dimer of the alpha-arylsulfanyl carbon radical). Whereas the fragmentation rate decreases as the C-C bond dissociation energy (BDE) increases, no rate change was observed by the replacement of OH by OD in the sulfide radical cation (k(OH)/k(OD) = 1). This suggests a transition state structure with partial C-C bond cleavage where the main effect of the OH group is the stabilization of the transition state by hydrogen bonding with the solvent. The fragmentation rate of 2-hydroxy sulfanyl radical cations turned out to be significantly slower than that of nitrogen analogues of comparable reduction potential, probably due to a more efficient overlap between the SOMO in the heteroatom and the C-C bond sigma-orbital in the second case. The fragmentation rates of 1(+*)-3(+*) were found to increase by addition of a pyridine, and plots of k(base) against base strength were linear, allowing calculation of the beta Bronsted values, which were found to increase as the reduction potential of the radical cation decreases, beta = 0.21 (3(+*)), 0.34 (1(+*)), and 0.48 (2(+*)). The reactions of 1(+*) exhibit a deuterium kinetic isotope effect with values that increase as the base strength increases: k(OH)/k(OD) = 1.3 (pyridine), 1.9 (4-ethylpyridine), and 2.3 (4-methoxypyridine). This finding and the observation that with the above three bases the rate decreases in the order 3(+*) > 1(+*) > 2(+*), i.e., as the C-C BDE increases, suggest that C-C and O-H bond cleavages are concerted but not synchronous, with the role of OH bond breaking increasing as the base becomes stronger (variable transition state). It is probable that, with the much stronger base, 4-(dimethylamino)pyridine, a change to a stepwise mechanism may occur where the slow step is the formation of a radical zwitterion that then rapidly fragmentates to products.
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http://dx.doi.org/10.1021/jo0486544DOI Listing
November 2004

Electron transfer and singlet oxygen mechanisms in the photooxygenation of dibutyl sulfide and thioanisole in MeCN sensitized by N-methylquinolinium tetrafluoborate and 9,10-dicyanoanthracene. The probable involvement of a thiadioxirane intermediate in electron transfer photooxygenations.

J Am Chem Soc 2003 Dec;125(52):16444-54

Dipartimento di Chimica, Università degli Studi di Roma La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.

Photooxygenations of PhSMe and Bu2S sensitized by N-methylquinolinium (NMQ+) and 9,10-dicyanoanthracene (DCA) in O2-saturated MeCN have been investigated by laser and steady-state photolysis. Laser photolysis experiments showed that excited NMQ+ promotes the efficient formation of sulfide radical cations with both substrates either in the presence or in absence of a cosensitizer (toluene). In contrast, excited DCA promotes the formation of radical ions with PhSMe, but not with Bu2S. To observe radical ions with the latter substrate, the presence of a cosensitizer (biphenyl) was necessary. With Bu2S, only the dimeric form of the radical cation, (Bu2S)2+*, was observed, while the absorptions of both PhSMe+* and (PhSMe)2+* were present in the PhSMe time-resolved spectra. The decay of the radical cations followed second-order kinetics, which in the presence of O2, was attributed to the reaction of the radical cation (presumably in the monomeric form) with O2-* generated in the reaction between NMQ* or DCA-* and O2. The fluorescence quenching of both NMQ+ and DCA was also investigated, and it was found that the fluorescence of the two sensitizers is efficiently quenched by both sulfides (rates controlled by diffusion) as well by O2 (kq = 5.9 x 10(9) M(-1) s(-1) with NMQ+ and 6.8 x 10(9) M(-1) s(-1) with DCA). It was also found that quenching of 1NMQ* by O2 led to the production of 1O2 in significant yield (PhiDelta = 0.86 in O2-saturated solutions) as already observed for 1DCA*. The steady-state photolysis experiments showed that the NMQ+- and DCA-sensitized photooxygenation of PhSMe afford exclusively the corresponding sulfoxide. A different situation holds for Bu2S: with NMQ+, the formation of Bu2SO was accompanied by that of small amounts of Bu2S2; with DCA, the formation of Bu2SO2 was also observed. It was conclusively shown that with both sensitizers, the photooxygenations of PhSMe occur by an electron transfer (ET) mechanism, as no sulfoxidation was observed in the presence of benzoquinone (BQ), which is a trap for O2-*, NMQ*, and DCA-*. BQ also suppressed the NMQ+-sensitized photooxygenation of Bu2S, but not that sensitized by DCA, indicating that the former is an ET process, whereas the second proceeds via singlet oxygen. In agreement with the latter conclusion, it was also found that the relative rate of the DCA-induced photooxygenation of Bu2S decreases by increasing the initial concentration of the substrate and is slowed by DABCO (an efficient singlet oxygen quencher). To shed light on the actual role of a persulfoxide intermediate also in ET photooxygenations, experiments in the presence of Ph2SO (a trap for the persulfoxide) were carried out. Cooxidation of Ph2SO to form Ph2SO2 was, however, observed only in the DCA-induced photooxygenation of Bu2S, in line with the singlet oxygen mechanism suggested for this reaction. No detectable amounts of Ph2SO2 were formed in the ET photooxygenations of PhSMe with both DCA and NMQ+ and of Bu2S with NMQ+. This finding, coupled with the observation that 1O2 and ET photooxygenations lead to different product distributions, makes it unlikely that, as currently believed, the two processes involve the same intermediate, i.e., a nucleophilic persulfoxide. Furthermore, the cooxidation of Ph2SO observed in the DCA-induced photooxygenation of Bu2S was drastically reduced when the reaction was performed in the presence of 0.5 M biphenyl as a cosensitizer, that is, under conditions where an (indirect) ET mechanism should operate. This observation confirms that a persulfoxide is formed in singlet oxygen but not in ET photosulfoxidations. The latter conclusion was further supported by the observation that also the intermediate formed in the reaction of thianthrene radical cation with KO2, a reaction which mimics step d (Scheme 2) in the ET mechanism of photooxygenation, is an electrophilic species, being able to oxidize Ph2S but not Ph2SO. It is thus proposed that the intermediate involved in ET sulfoxidations is a thiadioxirane, whose properties (it is an electrophilic species) seem more in line with the observed chemistry. Theoretical calculations concerning the reaction of a sulfide radical cation with O2-* provide a rationale for this proposal.
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http://dx.doi.org/10.1021/ja037591oDOI Listing
December 2003
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