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Caveolae associated proteins and how they effect caveolae dynamics
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Richard Lundmark)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Caveolae-associerade proteiner och hur dom påverkar dynamiken hos caveolae (Swedish)
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1668
Keyword [en]
Caveolae, caveolin, EHD2, cavin, microdomain, microscopy, TIRF, AFM
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-92500ISBN: 978-91-7601-114-0 (print)OAI: oai:DiVA.org:umu-92500DiVA: diva2:741088
Public defence
2014-09-19, N420, Naturvetarhuset, Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2014-08-29 Created: 2014-08-27 Last updated: 2014-08-29Bibliographically approved
List of papers
1. EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization
Open this publication in new window or tab >>EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization
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2012 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 23, no 7, 1316-1329 p.Article in journal (Refereed) Published
Abstract [en]

Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.

Place, publisher, year, edition, pages
American society for cell biology, 2012
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-55359 (URN)10.1091/mbc.E11-09-0787 (DOI)000302215400016 ()
Available from: 2012-05-31 Created: 2012-05-14 Last updated: 2017-12-07Bibliographically approved
2. Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2
Open this publication in new window or tab >>Structural insights into membrane interaction and caveolar targeting of dynamin-like EHD2
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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.

National Category
Cell Biology Biophysics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-88288 (URN)10.1016/j.str.2013.12.015 (DOI)000333025700007 ()24508342 (PubMedID)
Available from: 2014-05-05 Created: 2014-04-29 Last updated: 2017-12-05Bibliographically approved
3. An EH-domain switching mechanism regulates stable membrane association of EHD2
Open this publication in new window or tab >>An EH-domain switching mechanism regulates stable membrane association of EHD2
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

EHD2 is a dimeric ATPase known to stabilise the surface connection of the characteristic small invaginations of the cell surface termed caveolae. EHD2 oligomerises into rings around lipid membranes thereby controlling their shape. Here, we have analysed the domain interactions and mechanism that control the stable membrane association of EHD2 at caveolae. We have found that the N-terminus of EHD2, which is buried in the core protein and obstruct assembly, has to be relieved by an EH domain dependent mechanism. The binding between the EH domain and a loop in the GTPase domain of EHD2 was required for stable membrane association, but the loop in itself was not sufficient for specific recruitment to caveolae. A positively charged stretch in the EH domain is proposed to bind to lipids and thereby influence the exchange rate of EHD2. Taken together, we propose a stringent regulatory mechanism for the assembly of EHD2 involving switching of the EH domain position to release the N-terminus and facilitate oligomerisation.

National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-92494 (URN)
Available from: 2014-08-27 Created: 2014-08-27 Last updated: 2014-09-08Bibliographically approved
4. Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae
Open this publication in new window or tab >>Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae
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2015 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 5, 979-991 p.Article in journal (Refereed) Published
Abstract [en]

Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis due to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 is targeted to caveolae by cavin1 where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 region, showing that the cavins form distinct subcomplexes via their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae with short duration time at the membrane. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

Keyword
Cavin1, Cavin3, Caveolin1, Caveolae, EHD2
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-92497 (URN)10.1242/jcs.161463 (DOI)000350569900014 ()
Available from: 2014-08-27 Created: 2014-08-27 Last updated: 2017-12-05Bibliographically approved

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