Magnetogenetic Control of Cellular Functions using Biofunctionalized Magnetic Nanoparticles Inside Living Cells

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Title: Magnetogenetic Control of Cellular Functions using Biofunctionalized Magnetic Nanoparticles Inside Living Cells
Authors: Kappen, Marie
ORCID of the author:
Thesis advisor: Prof. Dr. Jacob Piehler
Thesis referee: Prof. Dr. Joost Holthuis, Ph.D.
Abstract: Remote control of cellular functions using magnetic forces offers unique opportunities in fundamental research and biomedical applications. The intracellular application of functionalized magnetic nanoparticles (MNP) provides the possibility to spatiotemporally increase the concentration of specific target proteins and thus locally enhance protein interactions (space mode magnetogenetic control). These can be exploited for site-specific activation of signaling pathways, for instance by increasing reaction turnovers or by inducing phase separation. However, designing MNPs suitable for effective, unbiased manipulation of target proteins inside cells has remained challenging. This work aimed to design biofunctionalization of MNPs for space mode magnetic manipulation of downstream signaling effector proteins at the plasma membrane and in the cytosol. For initial proof-of-concept experiments, application of previously established magnetic intracellular stealth MNPs based on natural ferritin (MagIcS) as a highly biocompatible protein cage were explored. As these exhibited limitations in terms of magnetic properties, a new MNP design was implemented based on a single-step surface coating of synthetic magnetic core nanoparticles with green fluorescent protein (GFP) fused to the iron binding site of Mms6 from magnetotactic bacteria (syMagIcS). In doing so, a stable biocompatible coating was created, which simultaneously enables site-specific recruitment of proteins of interest to the magnetic nanoparticle surface. The direct functionalization of synthetic magnetic cores yielded optimized magnetic properties and offers the possibility to customize core sizes and thus further enhance magnetic responsiveness. Exploiting the coherent functional design of MagIcS and syMagIcS, applicability for intracellular magnetogenetic use in space mode was explored at different levels. Efficient in situ MNP biofunctionalization with intracellular effector proteins by direct capturing via GFP nanobodies was achieved and intracellular translocation by using magnetic field gradients was obtained for both types of MNP. Activation of G-proteins at the plasma membrane was achieved by magnetogenetic translocation of MNP-bound catalytically active region of GEF proteins, with syMagIcS clearly showing superior performance as compared to MagIcS. Furthermore, liquid-liquid phase separation of the intrinsically disordered protein Dvl2, which plays an important role in Wnt signaling pathways, was induced by space mode magnetogenetics. With this tool at hand, we aim to understand the activation and regulatory mechanisms of these signaling pathways in more detail and test hypotheses by manipulating the signaling activation. These proof-of-concept experiments highlight the exciting possibilities of space mode magnetic manipulation to explore the spatiotemporal regulation of cellular processes in living cells and open new avenues in regenerative medicine.
Subject Keywords: Magnetogenetics; Magnetic Nanoparticles; Remote Control of Cellular Functions; small GTPases Rac1; wnt signaling pathway; small GTPases HRas; Biofunctionalization; Functional Coating; Magnetic Manipulation of Signaling Pathway
Issue Date: 5-May-2023
Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

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