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

dc.contributor.advisorSteinem, Claudia Prof. Dr.
dc.contributor.authorTeiwes, Nikolas
dc.format.extent186 (174)de
dc.titleGiant plasma membrane vesicle-derived in vitro systems to study the structure and dynamics of CaV1.3 channel clustersde
dc.contributor.refereeSteinem, Claudia Prof. Dr.
dc.description.abstractengThe 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 clustersde
dc.contributor.coRefereeMeinecke, Michael Prof. Dr.
dc.subject.engGiant Plasma Membrane Vesiclede
dc.subject.engSolid-supported Plasma Membranede
dc.subject.engVoltage-gated Calcium channel 1.3de
dc.subject.engPore-spanning Plasma Membranede
dc.subject.engsuper-resolution microscopyde
dc.subject.engFlorescence Microscopyde
dc.subject.engin vitro systemde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.notes.confirmationsentConfirmation sent 2023-11-06T19:45:01de

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