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Characterizing the cell cycle dependent regulation of adhesion to extracellular matrix proteins

English title Characterizing the cell cycle dependent regulation of adhesion to extracellular matrix proteins
Applicant Müller Daniel Jobst
Number 182587
Funding scheme Project funding (Div. I-III)
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Cellular Biology, Cytology
Start/End 01.05.2019 - 30.04.2023
Approved amount 1'119'000.00
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All Disciplines (2)

Discipline
Cellular Biology, Cytology
Biophysics

Keywords (25)

Cell Adhesion; Extracellular Matrix; Proteomics; Mechanotransduction; Single-Molecule Force Spectroscopy; Atomic Force Microscopy (AFM); Flow Cytometry; Confocal Microscopy; Crispr/Cas; Traction force microscopy; Single-Cell Force Spectroscopy; Integrins; Antibodies; Functional AFM imaging; Adhesome; Mitosis; Membrane Receptors; Extracellular Matrix ; Cell cycle; Adhesion formation; Super-resolution microscopy; Talin; Kindlin; Mitotic cell rounding; Cell cycle phases

Lay Summary (German)

Lead
Die Teilung adhärenter menschlicher und tierischer Zellen, geht mit der drastischen Veränderung der Zellform einher, in welcher sie für die Mitose eine nahezu perfekte Kugelform einnehmen. Um den präzise regulierten Prozess der mitotischen Zellrundung, durchzuführen, müssen Zellen ihre Adhäsion an ihre Umgebung der extrazellulären Matrix (ECM) regulieren. Hierzu wird vor allem die durch Integrine vermittelte Zelladhäsion aufgelöst und nach erfolgter Zellteilung wieder aufgebaut. Obwohl dieser für unsere Entwicklung, Gesundheit und Krankheit essentielle Vorgang bekannt ist, sind die Mechanismen, über welche mitotische Zellen ihre Adhäsion regulieren, weitgehend unverstanden.
Lay summary

In diesem Projekt untersuchen wir wie Zellen ihre durch Integrine vermittelte Adhäsion während des Zellzyklus regulieren. Integrine sind eine wichtige Familie von Membranrezeptoren welche tierische und menschliche Zellen nutzen, um an ihre extrazelluläre Matrix zu adhärieren. Um einen quantitativen Einblick in diesen wichtigen Vorgang des Lebens zu erhalten, wenden wir modernste nanotechnologische, mikroskopische und zellbiologische Methoden an. Dabei wird die Adhäsionskraft einzelner Zellen tierischen oder menschlichen Ursprungs gemessen, während die Prozesse der Zelle welche die Adhäsion regulieren sichtbar gemacht werden. Indem wir diese Prozesse abhängig vom Zellzyklus charakterisieren, liefern die Experimente Einsichten wie Zellen die Prozesse steuern. Wir erhoffen mit diesem Projekt neue und fundamentale Einsichten zu erzielen wie Zellen während des Zellzyklus ihre integrinabhängige Adhäsion steuern. Die Resultate werden eine direkte Auswirkung darauf haben wie Mediziner, Zellbiologen und Biotechnologen die Entwicklung von Geweben und Organen, die Zellmigration oder die Metastasierung von Tumoren neu betrachten und gegebenenfalls steuern können.

Direct link to Lay Summary Last update: 12.11.2018

Responsible applicant and co-applicants

Employees

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Number Title Start Funding scheme
160225 Charakterisierung der molekularen Mechanismen der a5ß1 and avß3 Integrin abhängigen Regulierung der Adhäsion von Fibroblasten 01.11.2015 Project funding (Div. I-III)
198231 Large-chamber high-resolution field-emission scanning electron microscope and focused ion beam system 01.06.2021 R'EQUIP
189807 Multiphoton Confocal Microscope for High-Speed and High-Resolution Imaging 01.09.2020 R'EQUIP

Abstract

In most adherent cells, mitosis is accompanied by drastic morphological changes, during which cells round to a nearly spherical shape. To facilitate this tightly regulated process, called mitotic cell rounding (MCR), cells need to disassemble integrin-mediated adhesion sites, which anchor them to the extracellular matrix (ECM). Integrins are a family of allosteric, heterodimeric adhesion receptors that connect distinct ECM proteins to the actin cytoskeleton via a plethora of intracellular adaptor and signaling proteins, which are collectively called ‘adhesome’. Cells employ adhesomes to sense and to respond to the biochemical and biophysical properties of the ECM. How cells disassemble adhesion sites to enable MCR, maintain basal adhesion to the ECM during mitosis and reestablish firm adhesion after cell division is unknown. With this proposed project, we aim to provide mechanistic insight on how cells regulate adhesion during the cell cycle, thereby focusing on adhesion regulation before, during and after cell division. Experimentally, we will quantify adhesion and traction forces during adhesion initiation, strengthening and maintenance in different cell cycle phases (G1/S/G2/M) using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS), traction force microscopy (TFM), multiparametric force-distance curve-based AFM (FD-based AFM) imaging, TIRF and confocal microscopy. In initial experiments, we found adhesion initiation to be compromised during mitosis. This may be linked to integrin conformations, which are fundamental to initiate adhesion. However, in which conformation, ranging from bent to fully extended, integrins bind ligands and recruit adhesome proteins is debated. We will thus use allosteric antibodies to stabilize integrins in distinct conformations and characterize their ligand-binding strengths and kinetics using SCFS, multiparametric AFM and TFM. Further, we will apply quantitative proteomics approaches to elucidate in which conformations integrins recruit adhesome proteins. This insight is particularly important because adhesome proteins affect integrin conformations, ligand-binding properties and adhesion reinforcement. Our preliminary data also shows that adhesion strengthening is impaired during mitosis suggesting for a failure of adhesion reinforcement by intracellular adaptor proteins. We will thus quantify the role of adhesome proteins on initiating and in reinforcing cell adhesion before, during and after mitosis. We will decipher the impact of the key adhesome proteins, including talin and kindlin, on initial cell adhesion. Since both proteins are required to form stable adhesions, we aim to decipher their synergistic role in integrin activation and adhesion reinforcement. Given the importance of adhesome proteins on adhesion regulation, we will also investigate which adaptor proteins are regulated to disassemble adhesions during MCR. Further, we will characterize whether and how the actin cytoskeleton, which strongly contracts during MCR, contributes to integrin-mediated adhesion disassembly. To gain mechanistic insights into adhesion regulation during the cell cycle, we will use cell lines depleted of distinct adhesome proteins, chemical perturb signaling proteins, and employ RNAi approaches in combination with SCFS, TFM, AFM and advanced optical microscopy. With the proposed project, we anticipate to contribute to a new and fundamental mechanistic understanding on how cells regulate integrins during the cell cycle. Furthermore, we will be able to translate our results on adhesion initiation to various other adhesion related fields, such as cell migration, tissue homeostasis, development and tumor metastasis.
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