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Mapping Rho GTPase signaling networks through acute, dynamic stimulation of spatio-temporal signaling fluxes.

English title Mapping Rho GTPase signaling networks through acute, dynamic stimulation of spatio-temporal signaling fluxes.
Applicant Pertz Olivier
Number 163061
Funding scheme Project funding (Div. I-III)
Research institution Institut für Zellbiologie Departement Biologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Cellular Biology, Cytology
Start/End 01.04.2016 - 31.05.2019
Approved amount 600'000.00
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Keywords (5)

Rho GTPase signaling; Chemical biology; Cytoskeleton; FRET biosensors; Live cell

Lay Summary (German)

Lead
Akute Perturbation Strategien um schnelle Spatio-temporal Signalisierungswege zu verstehen
Lay summary

 Während zelluläre Prozesse wie der Zellmigration wird das Zytoskeletts einer Zelle stetig auf Zeitskalen von Sekunden und Längenskalen einzelner Mikrometern remodelliert. Mit neuen Technologien können wir jetzt die Signaldynamik visualisieren, die dieses Zytoskelett reguliert. Diese Technologien haben neue Erkenntnisse möglich gemacht, die mit klassischen biochemischen Techniken, die nur den durschnittlichen Signalisierungszustand von tausenden von Zellen, mit einer schlechten Zeitauflösung messen, einfach nicht möglich sind. Wie zu erwarten, sieht man dann, dass diese Signalaktivitäten auf der gleiche Zeit/Längen Skala wie das Zytoskelett fluktuieren. Um diese schnelle Signalisierungswege zu studieren brauchen wir nicht nur Technologien um die Signaldynamik zu visualizieren, sondern auch um diese auf diesen genauen Zeit- und Längenskalen zu stören (z.B. hemmen spezifische Signalkomponenten). In diesem Projekt werden wir neue Technologien entwickeln, die es uns ermöglichen, spezifische Signalmoleküle auf Sekunden/Minuten schnellen Zeitskalen, und auf der Stufe von einzelnen Zellen, akut zu stören. Dies wird dann verwendet, um eine Reihe von Signalisierungskomponenten die wir vorher identifiziert haben, zu studieren. Dadurch werden neue Signalisierungskomponenten identifiziert, die spezifische Zytoskelett-Aktivitäten regulieren. Dies ergibt wesentliche neue Einblicke, wie präzise Signalmuster in einzelnen Zellen erzeugt werden. Dies könnte wichtige neue Erkenntnisse liefern, die es uns ermöglichen, aberrante Zellmigrationsprozesse in Pathologien wie metastasierendem Krebs oder Entzündungen zu verhindern.

 

Direct link to Lay Summary Last update: 07.03.2016

Responsible applicant and co-applicants

Employees

Collaboration

Group / person Country
Types of collaboration
Matthias Wymann/Dept. of Biomedicine/UniBas Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Noo Li Jeon, Seoul National University Korean Republic (South Korea) (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Gaudenz Danuser, UT Southwestern, Dallas United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
GCB Symposium 2019 Poster Signaling mechanisms of non-genetic cancer drug resistance in melanoma 30.01.2019 bern, Switzerland Mattei Alberto;
Goodbye Flat Biology: In Vivo inspired Cancer Biology and Therapy Poster ERK and Akt Signalling Dynamics in 3D Organoid Models of Breast Cancer - Associated Mutations 09.09.2018 Berlin, Germany Ender Pascal;
BeFri Research Retreat 2018 Poster ERK and Akt Signalling Dynamics in 3D Organoid Models of Breast Cancer - Associated Mutations 26.04.2018 Kandersteg, Switzerland Ender Pascal;
International Symposium Measuring and Modelling Cell Migration Poster Using acute perturbation techniques to dissect spatio-temporal Rho GTPase signaling networks 22.02.2018 Vienna, Austria van Unen Jacobus; Pertz Olivier;
GCB Symposium 2018 Poster Signaling mechanisms of non-genetic cancer drug resistance in melanoma 01.02.2018 Bern, Switzerland Mattei Alberto;
GCB Symposium 2018 Poster Understanding Single Cell - Level MAPK Activation Dynamics for Manipulation of Neuronal Stem Cell Self - Renewal and Differentiation Fates 01.02.2018 Bern, Switzerland Ender Pascal; Pertz Olivier;
BeFri Research Retreat Poster Signaling mechanisms of non-genetic cancer drug resistance in melanoma 04.05.2017 Kandersteg, Switzerland Mattei Alberto;


Associated projects

Number Title Start Funding scheme
173462 Dissecting a Rho GTPase spatio-temporal signaling network regulating growth cone motility, neurite and axonal outgrowth 01.11.2017 Bilateral programmes
149923 Optogenetic control of receptor tyrosine kinase signaling to manipulate cell fate 01.02.2014 Project funding (Div. I-III)
177096 A high content confocal microscope for fast 3D imaging of living samples 01.10.2018 R'EQUIP

Abstract

Rho GTPases are signaling molecules that regulate cytoskeletal dynamics. Current models state that each GTPase regulates one specific cytoskeletal polymer: Rac1 controls membrane protrusion, Cdc42 controls filopodium formation, and RhoA regulates contractility. Recent developments in technologies to visualize Rho GTPase spatio-temporal activation dynamics have however revealed a much more complex picture. During fibroblast leading edge protrusion, or neuronal growth cone motility, RhoA, Rac1 and Cdc42 are all activated in micrometer-sized, overlapping subcellular domains. Furthermore, their activity fluctuates on time scales of tens of seconds. Such a complex spatio-temporal signaling program most likely serves to precisely position specific cytoskeletal regulating activities to fine tune these morphogenetic events. Understanding this signaling complexity requires a systematic approach to answer two fundamental questions: 1. What are the mechanisms that shape the specific subcellular Rho GTPase activation patterns in time and space ? 2. What is the function of each Rho GTPase with respect to cytoskeletal regulation ?This proposal builds on novel technologies and conceptual frameworks our lab has recently evolved. Specifically, we have: 1. built 2nd generation, improved RhoA, Rac1 and Cdc42 FRET-based biosensors; 2. performed initial characterization of the function of all Rho GTPase interactors (upstream regulators such as GEFs and GAPs, downstream effectors) during growth cone motility; 3. set up an automated growth cone segmentation computer vision pipeline to characterize their dynamics. One important problem we are currently facing is that “long term application” of any molecular perturbation (by example by means of RNAi-mediated knockdown), alters cell morphology and morphodynamics, affecting mechanical feedbacks that impinge on spatio-temporal control of Rho GTPases. This precludes a clear understanding of the causal events that leads to altered Rho GTPase regulation in response to a molecular perturbation.Here, by integrating genome editing and chemical biology techniques, we propose to solve this very specific issue, by evolving a technology that acutely applies the perturbation at the same temporal scale than Rho GTPase signaling fluctuates. Using this technology, we will systematically perturb a panel of endogenous Rho GTPase regulators (GEFs and GAPs) on time scales of tens of seconds. We will then record Rho GTPase activation dynamics (using FRET biosensors), as well as downstream cytoskeletal dynamics in response to these perturbations during both neuronal growth cone and fibroblast motility model systems. Our computer vision algorithms will be used to quantify the cellular responses to these perturbations. By answering the two questions mentioned at the beginning of this summary, our integrated approach has the potential to unambiguously deconvolve a spatio-temporal Rho GTPase signaling network at systems biology level. An important component of such a systems biology level understanding, is that it might indicate emergent properties that allow for robust manipulation of cell migration or growth cone motility for biomedical purposes.
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