Giant plasma membrane vesicle-derived in vitro systems to study the structure and dynamics of CaV1.3 channel clusters

Teiwes, Nikolas

dc.contributor.advisor	Steinem, Claudia Prof. Dr.
dc.contributor.author	Teiwes, Nikolas
dc.date.accessioned	2023-11-06T16:55:24Z
dc.date.available	2024-10-04T00:50:14Z
dc.date.issued	2023-11-06
dc.identifier.uri	http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14956
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-10173
dc.format.extent	186 (174)	de
dc.language.iso	eng	de
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/
dc.subject.ddc	571.4	de
dc.title	Giant plasma membrane vesicle-derived in vitro systems to study the structure and dynamics of CaV1.3 channel clusters	de
dc.type	doctoralThesis	de
dc.contributor.referee	Steinem, Claudia Prof. Dr.
dc.date.examination	2023-10-05	de
dc.description.abstracteng	The voltage-gated calcium channel 1.3 (CaV1.3) has plentiful tasks in the human body. One of them is the excitation-contraction coupling within the heart, where it is known to form CaV1.3 channel cluster. However, their role is not clearly understood, and an appropriate investigative system is lacking. To develop a sophisticated system, novel vesiculation agents were added to mammalian cells to generate giant plasma membrane vesicles (GPMVs). GPMVs display a reduced cellular complexity which allows for more precise investigations on fundamental plasma membranes properties and its components. By implementing a gentle GPMV spreading protocol based on silanol-functionalization techniques, solid supported plasma membrane bilayers (SPMBs) and pore-spanning plasma membranes (PSPMs) were obtained. These plasma membrane systems provided useful insights on properties. Membrane structure, phase behavior, lateral diffusion, and membrane viscosity were investigated using various bioimaging techniques such as confocal and atomic force as well as stimulated excitation depletion (STED) microscopy. Exploiting cell-derived systems on a heterologous expression system for CaV1.3, a transfer of CaV1.3 to the SPMBs system could be achieved. Utilizing a HaloTag labeling strategy on CaV1.3 channel (Halo-CaV1.3), Halo-CaV1.3 channel cluster (Halo-CaV1.3 cluster) were resolved on SPMBs by STED microscopy, resulting in significantly improved resolution compared to live cells. The HaloTag strategy allowed us to count individual channels and determine the density of clusters. As a result, insights on endosomal CaV1.3 cluster trafficking and stimulated β-adrenergic response of the CaV1.3 cluster were analyzed in a high-throughput format. These findings contribute to an understanding of CaV1.3 cluster structure and suggest a loose assembly of CaV1.3 channels supported by scaffolding proteins, with cluster density gradually increasing at a higher channel number. In addition, CaV1.3 cluster were also analyzed in free-standing PSPMs, approving via observed Halo-CaV1.3 cluster dynamics previous statements on CaV1.3 cluster assembly. During this research GPMV-derived in vitro systems have proven to be a versatile tool to study structure and dynamics of CaV1.3 channel clusters	de
dc.contributor.coReferee	Meinecke, Michael Prof. Dr.
dc.subject.eng	GPMV	de
dc.subject.eng	Giant Plasma Membrane Vesicle	de
dc.subject.eng	CaV1.3	de
dc.subject.eng	Solid-supported Plasma Membrane	de
dc.subject.eng	SPMB	de
dc.subject.eng	Voltage-gated Calcium channel 1.3	de
dc.subject.eng	PSPM	de
dc.subject.eng	Pore-spanning Plasma Membrane	de
dc.subject.eng	super-resolution microscopy	de
dc.subject.eng	Florescence Microscopy	de
dc.subject.eng	in vitro system	de
dc.identifier.urn	urn:nbn:de:gbv:7-ediss-14956-2
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.description.embargoed	2024-10-04	de
dc.identifier.ppn	1871222265
dc.identifier.orcid	0009-0004-3944-0801	de
dc.notes.confirmationsent	Confirmation sent 2023-11-06T19:45:01	de

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GGNB - Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [1169]
GGNB - Göttingen Graduate Center for Neurosciences, Biophysics and Molecular Biosciences

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