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Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Richard Lundmark)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Richard Lundmark)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Richard Lundmark)
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Richard Lundmark)
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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.

Place, publisher, year, edition, pages
2015. Vol. 128, no 5, 979-991 p.
Keyword [en]
Cavin1, Cavin3, Caveolin1, Caveolae, EHD2
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-92497DOI: 10.1242/jcs.161463ISI: 000350569900014OAI: oai:DiVA.org:umu-92497DiVA: diva2:741056
Available from: 2014-08-27 Created: 2014-08-27 Last updated: 2017-12-05Bibliographically 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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1668
Keyword
Caveolae, caveolin, EHD2, cavin, microdomain, microscopy, TIRF, AFM
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
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)
Opponent
Supervisors
Available from: 2014-08-29 Created: 2014-08-27 Last updated: 2014-08-29Bibliographically approved
2. Regulation of assembly and cell surface dynamics of caveolae
Open this publication in new window or tab >>Regulation of assembly and cell surface dynamics of caveolae
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A typical mammalian cell plasma membrane displays a high level of plasticity counter balanced with stability. The plasma membrane show various kinds of invaginations to meet physiological demands of the cells such as nutrient uptake, receptor signaling etc. An example of one such invagination observed in many cell types is “Caveola”. Caveolae are bulb shaped invaginations of the plasma membrane enriched in sphingolipids and cholesterol. Each individual caveola is in equilibrium with multi caveolar assemblies and exhibits various dynamics ranging from stable association with cell surface to the “kiss and run” type and internalized caveolae called cavicle. The principle protein components of caveolae involve caveolin1, 2 and 3, cavin 1, 2, 3 and 4 along with EHD2, PACSIN2 and dynamin. Caveolin1 acts as the hallmark of caveolae, whereas caveolin3 and cavin4 are limited to muscle cells. Caveolae are appreciated as important plasma membrane structures in maintaining cellular homeostasis of many cell types. Its dysfunction is associated with several human diseases such as cancer, vascular diseases, lipid and muscular dystrophies.

This thesis aims to understand the assembly of caveolae and the molecular machineries involved in the regulation of caveolae dynamics. In particular, we have focused on the mechanism of cavin coat assembly, the influence of cavin3 in the regulation of caveolae dynamics, along with the mechanistic cycle of EHD2.

Cavins form characteristic striations around caveolae, and in this work we showed that the N-terminus of cavin3 interacts with the trimeric N-terminus of cavin1 in competition with the N-terminus of cavin2 to form cavin sub-complex. We also observed that cavin3 interacts with caveolin1 in a cholesterol dependent manner and cavin3 may promote scission by acting as a positive regulator of caveolae dynamics, opposite to the cellular function of EHD2. The stringent roles of cavin3 and EHD2 control the equilibrium between stably cell surface associated caveolae and the “kiss and run” type of caveolae, undergoing rounds of fission and fusion. Our results demonstrated the molecular composition of the caveolae coat at a domain level with the stoichiometry of cavin sub-complexes. We also showed the function of cavin3 in the regulation of caveolae dynamics at the plasma membrane. Previous work from our lab showed that EHD2 is a dimeric ATPase localised to the caveolae neck and confines caveolae to the cell surface. In the present study, we showed that EHD2 oligomerized in an open conformation stabilized by ATP and in a G-domain loop dependent manner. The oligomerization of EHD2 in cells is finely tuned by the N-terminal region and the C-terminal EH domain, where both of these regions act as negative regulators of membrane binding. Our results showed the stringent regulation of EHD2 oligomerization and its importance with respect to various statuses and/or function of caveolae.

In summary, the current study provides a novel insight into the assembly of cavin coat and protein machineries involved in the regulation of caveolae dynamics. In addition, it also contributes to the understanding of the molecular mechanism of ATPase activity of EHD2. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 52 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1816
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-120753 (URN)978-91-7601-496-7 (ISBN)
Public defence
2016-06-15, N450, Naturvetarhuset, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-24 Created: 2016-05-20 Last updated: 2016-05-26Bibliographically approved

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Mohan, JaganMorén, BjörnLarsson, ElinHolst, MikkelLundmark, Richard
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