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Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2014 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 22, no 3, 409-420 p.Article in journal (Refereed) Published
Abstract [en]

The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.

Place, publisher, year, edition, pages
2014. Vol. 22, no 3, 409-420 p.
National Category
Cell Biology Biophysics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
URN: urn:nbn:se:umu:diva-88288DOI: 10.1016/j.str.2013.12.015ISI: 000333025700007PubMedID: 24508342OAI: diva2:715470
Available from: 2014-05-05 Created: 2014-04-29 Last updated: 2014-09-08Bibliographically approved
In thesis
1. Caveolae associated proteins and how they effect caveolae dynamics
Open this publication in new window or tab >>Caveolae associated proteins and how they effect caveolae dynamics
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Caveolae-associerade proteiner och hur dom påverkar dynamiken hos caveolae
Abstract [en]

Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae.

We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells.

In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface.

In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 54 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1668
Caveolae, caveolin, EHD2, cavin, microdomain, microscopy, TIRF, AFM
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry
urn:nbn:se:umu:diva-92500 (URN)978-91-7601-114-0 (ISBN)
Public defence
2014-09-19, N420, Naturvetarhuset, Umeå Universitet, Umeå, 10:00 (English)
Available from: 2014-08-29 Created: 2014-08-27 Last updated: 2014-08-29Bibliographically approved

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Morén, BjörnLundmark, Richard
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