Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Cell Scientists to Watch
    • First Person
    • Sign up for alerts
  • About us
    • About JCS
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Fast-track manuscripts
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • JCS Prize
    • Manuscript transfer network
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JCS
    • Subscriptions
    • Advertising
    • Feedback
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Journal of Cell Science
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Journal of Cell Science

  • Log in
Advanced search

RSS   Twitter  Facebook   YouTube  

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Cell Scientists to Watch
    • First Person
    • Sign up for alerts
  • About us
    • About JCS
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Fast-track manuscripts
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • JCS Prize
    • Manuscript transfer network
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JCS
    • Subscriptions
    • Advertising
    • Feedback
Research Article
Evolutionary analysis and molecular dissection of caveola biogenesis
Matthew Kirkham, Susan J. Nixon, Mark T. Howes, Laurent Abi-Rached, Diane E. Wakeham, Michael Hanzal-Bayer, Charles Ferguson, Michelle M. Hill, Manuel Fernandez-Rojo, Deborah A. Brown, John F. Hancock, Frances M. Brodsky, Robert G. Parton
Journal of Cell Science 2008 121: 2075-2086; doi: 10.1242/jcs.024588
Matthew Kirkham
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan J. Nixon
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mark T. Howes
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Laurent Abi-Rached
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Diane E. Wakeham
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Hanzal-Bayer
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Charles Ferguson
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michelle M. Hill
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Manuel Fernandez-Rojo
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Deborah A. Brown
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John F. Hancock
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Frances M. Brodsky
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert G. Parton
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & tables
  • Supp info
  • Info & metrics
  • PDF + SI
  • PDF
Loading

Data supplements

  • JCS024588 Supplementary Material

    Files in this Data Supplement:

    • Supplemental Figure S1 (Adobe PDF) -

      Fig. S1. Alignment of caveolin sequences used to generate phylogenetic trees. Residues 54-158 from human Cav1 (HsCav1) were aligned with the corresponding residues of the indicated species. The residue numbers for HsCav1 are used throughout the text. The other numbering systems (denoted FMB and alignment) were used during data analysis. Latin species names are indicated with the genus abbreviated by a single letter. Residues highlighted in grey are conserved in &γτ;90% of all sequences and residues collared in red are only conserved in caveolin sequences that can form caveolae. Sequences are named as described for Fig 1. Gaps in alignment are noted as −. Mutations or deletions of Cav3 found in human muscle diseases are denoted by either black asterisks or delta symbols, respectively. Triple amino-acid deletions of Cav3 are denoted by a line. Cav1 mutations involved in human disease are denoted by red asterisks. palmitoylated cysteine residues are denoted by +.

    • Supplemental Figure S2 (Adobe PDF) -

      Fig. S2. Tubular structures formed in cells expressing flotillin-1 and flotillin-2. Cav1-null MEFs were transiently transfected with flotillin-1-GFP and flotillin-2-RFP. Cells were FAC sorted, fixed and examined by electron microscopy. No caveolae-like structures (as determined by morphology analysis) were observed in transfected cells, but an increase in tubular structures was observed.

    • Supplemental Figure S3 (Adobe PDF) -

      Fig. S3. CeCav-a does not behave in a manner similar to HsCav1 in mammalian cells. (A-C) Microinjection of Cav1-null MEFs was used to cause the overexpression of either HsCav1-HA (A,B), CeCav-a-flag (A,C). Cholera toxin binding (CTB) subunit was bound to the surface of cells, then incubated at 37°C for 40 minutes and fixed and processed for immunofluorescence confocal microscopy using antibodies against GM130 and either the HA epitope or the FLAG epitope. In control Cav1-null MEFs, CTB accumulated in a GM130-positive compartment, judged as the Golgi complex (A). In cells overexpressing HsCav1-HA, CTB accumulation in the Golgi complex was reduced (asterisks), as compared with neighbouring control cells. Overexpression of CeCav-a-FLAG did not affect CTB accumulation in the Golgi complex. The fluorescence intensity of CTB accumulation in the Golgi complex was measured in injected and non-injected cells (graphs B and C). There was a significant reduction in the accumulation of CTB in the Golgi complex in cells over expressing HsCav1-HA, although no significant difference in cells injected with CeCav-a-Flag as compared to controls. (**P<0.001). (D-E) Cav1-null MEFs expressing HsCav1-HA (D,F) or CeCav-a-HA (E) were fixed, incubated with anti-HA antibodies, and processed for TIRF microscopy. HsCav1-HA appeared in defined punctate structures within the plasma membrane (see inset). CeCav-a-HA had a more diffuse irregular distribution within the plasma membrane than HsCav1-HA Scale bars: 10 µm; 5µm (inset). The frequency at which defined values of fluorescent intensity of the HsCav1-HA punctate occurred were plotted (F). HsCav1-HA puncta can be classified into groups according to their fluorescent intensity. The fluorescent intensities of the group increase in a defined quantal manner. A circular mask of fixed size was used to select 135 individual puncta, and the average intensity of these regions was recorded. The frequency of an intensity was calculated by creating pools (0.01 to 100, 100.01 to 200.01 etc..) from 0 to 4000. HsCav1-HA clearly exhibited punctate fluorescent structures of defined quantal units that correspond to either single caveola or double caveolar structures, unlike previous reports we were unable to identify multiple caveolae structures. This maybe due to the use of both primary and secondary antibodies to detect Cav1-HA compared with analysing Cav1-GFP (Pelkmans and Zerial, 2005) where the fluorescent signal may be a more linear representation of the amount of Cav1.

    • Supplemental Figure S4 (Adobe PDF) -

      Fig. S4. CeCav-a does not behave in a manner similar to HsCav1 in mammalian cells − continued analysis. (A) Confluent BHK cells transfected with HsCav1-HA and CeCav-a-Flag, were allowed to recover for 5 hours after the cell monolayer was wounded. Cells were fixed, permeablised and probed with anti-FLAG (red) and anti-HA (green) antibodies before being processed for immunofluorescent microscopy. The wound in the cell monolayer was analysed for polarised migrating cells, an arrow shows direction of cell migration and the cell has been outline in red. (B) Cav1-null MEFs transiently expressing HsCav1-YFP or CeCav-a-Flag, were incubated with oleic acid for 15 hours and then processed for immunofluorescence microscopy. Cells were labelled with the neutral lipid dye, Oil Red. All cells examined had accumulated Oil Red-positive lipid droplets. Oleic acid treatment caused a redistribution of HsCav1-YFP to lipid droplets but did not affect CeCav-a localisation (see insets). (C) Cav1-null MEFs co-expressing Rab5Q79L and either HsCav1 or CeCav-a. CeCav-a, but not HsCav1, accumulates in Rab5Q79L-enlarged early endosomes. Scale bars: 10 µm (A), 20µm (B,C), 10 µm (insets).

    • Supplemental Figure S5 (Adobe PDF) -

      Fig. S5. Analysis of mutant caveolin proteins using a combination of light- and electron-microscopy assays. (A) Cav1-null MEFs expressing truncation mutants of CeCav-a and hybrid proteins between HsCav1 and CeCav-a were either fixed and directly processed, or incubated with anti-HA antibodies, before being processed for confocal microscopy. HCav-HA49-132, HCav-HA1-132 and HCav-HACeCav1-110 all had a distribution similar to Cav1 in WT MEFs and formed caveolae. HCav-HA102-132, HCav-HA80-132 and HCav-cherry60-132 were localised to the plasma membrane but failed to form caveolae as determined by EM assay. CeCav-a-HAΔ1-86 was localised to intracellular vesicular. HCav1-101 was restricted to a perinuclear compartment. The results of the EM caveolae biogenesis assay are indicated in the table below. (B) Cav1-null MEFs transiently expressing Cav-HA trunctation mutants were fixed and labelled with anti-HA antibodies and examined by confocal microscopy. The transiently expressed deletion mutants of CfCav1-HAΔ157-178, CfCav1-HAΔ147-178 and MmCav3 Δ 1-16 were localised to the PM and formed caveolae. All other constructs had a restricted perinuclear localisation resembling the Golgi complex. The presence or absence of caveolae according to the results of the EM caveola biogenesis assay are also indicate. (C) Cav1-null MEFs expressing CfCav1-HACYS-, HsCav1-HAS80A, HsCav1-HAS80E, were fixed and incubated with anti-HA antibodies, before being processed for confocal microscopy or processed for EM caveolae biogenesis assay. CfCav1-HACYS- and HsCav1-HAS80A had a distribution similar to Cav1 in WT MEFs and formed caveolae. Cav1-HAS80E was restricted to a perinuclear compartment. The results of the EM caveolae biogenesis assay are indicated in the table below. −, experiment was not performed. Scale bars: 10 µm; inset, 5µm.

    • Supplemental Figure S6 (Adobe PDF) -

      Fig. S6. Additional analysis of CeCav-a and the interaction of HCav constructs and Cav2. (A) Electron micrograph of Cav1-null MEFs expressing HCav1-101. Injected cells were identified by electron-dense nucleus (N+) as compared to non-injected cells (N-). No CTB-HRP-positive surface-connected flask- shaped or vesicular structures 50 nm in diameter were observed. Scale bar 5µm. (B) Cav1-null MEFs expressing HsCav1-HA, HCav1-101-HA, and HCav-cherry60-132 were fixed and, where appropriate, incubated with anti-HA antibodies and processed for TIRF microscopy. HsCav1-HA appeared in defined punctate structures within the PM, but both HCav1-101 and HCav60-132 had more irregular evenly distribution PM localisation. Only in a subset of cells was HCav1-101 localised to the PM. Scale bars 10 µm. (C) Cav1-null MEFs were transiently transfected with HsCav1-HA, HCav1-HA102-132, HCav1-HA80-132 and HCav1-cherry60-132. Localisation of Cav2 within these cells was investigated by immunofluorescence microscopy. Cells were probed with antibodies against Cav2 and HA. In untransfected Cav1-null MEFs Cav2 was retained in a perinuclear compartment. Cav1-HA and the hybrid proteins examined caused a redistribution of Cav2 from the Golgi complex even through they cannot form caveolae. Scale bars: 10 µm.

    • Supplemental Table S1 -

      Adobe PDF

    • Supplemental Table S2 -

      Adobe PDF

Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

 Download PDF

Email

Thank you for your interest in spreading the word on Journal of Cell Science.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Evolutionary analysis and molecular dissection of caveola biogenesis
(Your Name) has sent you a message from Journal of Cell Science
(Your Name) thought you would like to see the Journal of Cell Science web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Research Article
Evolutionary analysis and molecular dissection of caveola biogenesis
Matthew Kirkham, Susan J. Nixon, Mark T. Howes, Laurent Abi-Rached, Diane E. Wakeham, Michael Hanzal-Bayer, Charles Ferguson, Michelle M. Hill, Manuel Fernandez-Rojo, Deborah A. Brown, John F. Hancock, Frances M. Brodsky, Robert G. Parton
Journal of Cell Science 2008 121: 2075-2086; doi: 10.1242/jcs.024588
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Research Article
Evolutionary analysis and molecular dissection of caveola biogenesis
Matthew Kirkham, Susan J. Nixon, Mark T. Howes, Laurent Abi-Rached, Diane E. Wakeham, Michael Hanzal-Bayer, Charles Ferguson, Michelle M. Hill, Manuel Fernandez-Rojo, Deborah A. Brown, John F. Hancock, Frances M. Brodsky, Robert G. Parton
Journal of Cell Science 2008 121: 2075-2086; doi: 10.1242/jcs.024588

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article navigation

  • Top
  • Article
    • Summary
    • Introduction
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & tables
  • Supp info
  • Info & metrics
  • PDF + SI
  • PDF

Related articles

Cited by...

More in this TOC section

  • Calcium elevations disrupt interactions between intraflagellar transport and the flagella membrane in Chlamydomonas
  • SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72
  • EFA6A, an exchange factor for Arf6, regulates early steps in ciliogenesis
Show more RESEARCH ARTICLE

Similar articles

Other journals from The Company of Biologists

Development

Journal of Experimental Biology

Disease Models & Mechanisms

Biology Open

Advertisement

2020 at The Company of Biologists

Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.


Mole – The Corona Files

"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.


Cell scientist to watch – Christine Faulkner

In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.


Read & Publish participation extends worldwide

“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”

Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.


JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Special issues
  • Subject collections
  • Interviews
  • Sign up for alerts

About us

  • About Journal of Cell Science
  • Editors and Board
  • Editor biographies
  • Travelling Fellowships
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Fast-track manuscripts
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access
  • JCS Prize
  • Manuscript transfer network
  • Biology Open transfer

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contacts

  • Contact JCS
  • Subscriptions
  • Advertising
  • Feedback

Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992