Dataset Information

Versatile membrane deformation potential of activated pacsin.

ABSTRACT: Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pacsin isoform 1 (pacsin-1) has reduced activity compared to its isolated F-BAR domain, implicating an inhibitory role for its C-terminal Src homology 3 (SH3) domain. Here we show that the autoinhibitory, intramolecular interactions in pacsin-1 can be released upon binding to the entire proline-rich domain (PRD) of dynamin-1, resulting in potent membrane deformation activity that is distinct from the isolated F-BAR domain. Most strikingly, we observe the generation of small, homogenous vesicles with the activated protein complex under certain experimental conditions. In addition, liposomes prepared with different methods yield distinct membrane deformation morphologies of BAR domain proteins and apparent activation barriers to pacsin-1's activity. Theoretical free energy calculations suggest bimodality of the protein-membrane system as a possible source for the different outcomes, which could account for the coexistence of energetically equivalent membrane structures induced by BAR domain-containing proteins in vitro. Taken together, our results suggest a versatile role for pacsin-1 in sculpting cellular membranes that is likely dependent both on protein structure and membrane properties.

SUBMITTER: Goh SL

PROVIDER: S-EPMC3517540 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Versatile membrane deformation potential of activated pacsin.

Goh Shih Lin SL Wang Qi Q Byrnes Laura J LJ Sondermann Holger H

PloS one 20121207 12

Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pa ...[more]

PMID: 23236520

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Project description:Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (Vm). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring Vm signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map Vm and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan- and blue-elicited fluorescence have different sensitivities to Vm, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving Vm (excitation ratiometry). Reconstructed Vm signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map Vm and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics.

Dataset Information

Versatile membrane deformation potential of activated pacsin.

Publications

Versatile membrane deformation potential of activated pacsin.

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