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First published online 27 May 2008
doi: 10.1242/jcs.024588

Journal of Cell Science 121, 2075-2086 (2008)
Published by The Company of Biologists 2008
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Evolutionary analysis and molecular dissection of caveola biogenesis

Matthew Kirkham1,2, Susan J. Nixon1,2,*, Mark T. Howes1,2,*, Laurent Abi-Rached3,*, Diane E. Wakeham4, Michael Hanzal-Bayer1, Charles Ferguson1,2, Michelle M. Hill1, Manuel Fernandez-Rojo1,2, Deborah A. Brown5, John F. Hancock1, Frances M. Brodsky4 and Robert G. Parton1,2,{ddagger}

1 Institute for Molecular Bioscience, University of Queensland, Queensland 4072, Brisbane, Australia
2 Centre for Microscopy and Microanalysis, University of Queensland, Queensland 4072, Brisbane, Australia
3 Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
4 The G.W. Hooper Foundation, Departments of Biopharmaceutical Sciences, Pharmaceutical Chemistry and Microbiology and Immunology, University of California, San Francisco, CA 94143-0552, USA
5 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA


Figure 1
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  		Fig. 1. Evolutionary conservation of caveolins. Caveolin phylogeny. Phylogenetic analyses were performed with Bayesian interference, ML, NJ and parsimony methods from the alignment of caveolin sequences to R54 to P158 in HsCav1 to P158. The ML tree topology was used for the display (with a midpoint rooting) and the node support is given in the following order (from top to bottom): Bayesian (posterior probability, PP), ML, NJ and parsimony (bootstrap support, BS). Asterisks (*) indicate a PP<95 or a BS<50. Prot, protostomians. To simplify the display, several nodes with a good support (PP>95 and BS>=80) are marked by a filled circle; similarly the support for poorly supported nodes or for a few terminal nodes with average support was omitted. For the roots of the `CavY extended' and `Prot G1' groups the node support is indicated for two datasets, with the left-hand column derived from the complete dataset and the right-hand column derived from a reduced dataset where the two nematode sequences of the `Prot G3' group were removed. For the root of the `CavY extended' group the BS with the NJ analysis was <50 and the support with the Interior Branch Test is indicated in parentheses; the support on the left was for a `CavY extended' group including the two nematode sequences of the `Prot G3' group. The names of the species are colour-coded as indicated in the top-right corner of the figure.

 

Figure 2
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  		Fig. 2. Genomic organisation of caveolin genes and a model for the diversification of the caveolin sequences in metazoans. (A) Genomic localisation of caveolin-encoding genes in four species. Related families are indicated by related colours: Cav1 and Cav3 are pink, Cav2 and Cav2R are green and olive, respectively, and CavY-related sequences are yellow. When two genes are in a contiguous genomic segment the transcription orientation is indicated by an arrow above each gene. The symbol `//' between Cav2R and Cav3 in Tetraodon nigroviridis indicates a long genomic segment of ~1.3 Mb. (B) This model is based on the results of the phylogenetic analysis presented in Fig. 1 and summarises the interpretation in the text. For the parts of the model for which there is no clear phylogenetic support (represented by dotted lines), the simplest evolutionary model was selected based on the caveolin gene content information in the species studied and the ability of these sequences to form caveolae structures. For the CavY group, two levels of phylogenetic support are indicated: `minimum' (supported by all the phylogenetic analysis methods used) and `extended' (supported by two to three of the four methods used).

 

Figure 3
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  		Fig. 3. Analysis of the de novo formation of caveolae in mammalian cells. (A) Wild-type MEFs were labelled with anti-Cav1 (green) and anti-GM130 (red) antibodies and prepared for confocal microscopy or surface-labelled with CTB-HRP and prepared for electron microscopy. (B-E) Using identical methodology, Cav1-null MEFs injected with HRP and either HsCav1-HA (B), MmCav3-HA(C) AmCav-HA DNA (D) and CeCav-a-HA (E) and were prepared for both confocal and electron microscopy. All transiently expressed constructs, except CeCav-a-HA had the same subcellular distribution as Cav1 in wild-type MEFs as determined by confocal microscopy and produced surface-connected caveolae as identified with electron microscopy. Injected cells were identified by electron-dense HRP-DAB labelling of the nucleus. Scale bars: 10 µm (confocal images), 200 nm (EM images).

 

Figure 4
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  		Fig. 4. Quantification of the de novo formation of caveolae in mammalian cells. (A-F) Cav1-null MEFs transiently transfected with CfCav1-YFP (A,F), GFP (B,F), HsCav1β-GFP (C,F) CeCav-a-GFP (D,F), HCav49-132-GFP (F) or co-transfected with flotillin1-GFP and flotillin2-RFP (E,F), were FAC sorted by fluorescent intensity into two pools with different expression levels with the exception of flotillin-1 and flotillin-2. The cells were fixed and examined by electron microscopy. Caveolae (arrows) were only observed in cells transfected with CfCav1-GFP, HsCav1β-GFP and at a lower frequency in cells expressing HCav49-132-GFP. Clathrin-coated pits are highlighted by arrowheads. Scale bars: 200 nm. (F) Caveolae-like structures as determined by morphology were counted at the plasma membrane and the density of caveolae-like structures was determined. **P<=0.05, difference between high and low expression pools of the same construct.

 

Figure 5
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  		Fig. 5. Summary of the 33 different caveolin constructs analysed. (A) EM micrographs of Cav1-null MEFs microinjected with HRP and either HsCav-HA{Delta}3-48, MmCav3-HA{Delta}1-16, CfCav1-HA{Delta}147-178 or HCav-HA49-132 DNA. The cell surface was labelled with CTB-HRP. All the constructs generated caveolae that were similar to endogenous caveolae in wild-type MEFs. Scale bars: 200 nm. (B) Schematic protein alignment illustrating the protein-domain structure and some of the known post translational modifications of HsCav1, HsCav3 and CeCav-a. The scale bar indicates the number of amino acids from the start of the N-terminus of CeCav-a. Pink circles highlight palmitoylated residues, yellow circles mark some of the residues mutated in HsCav3 in muscle diseases. Circles with broken lines and lightly shaded mark non-conserved residues. (C) Summary of the results of EM and light-microscopy experiments from the constructs tested in this study. #, constructs that formed caveolae but were less efficient than wild-type constructs. ^, 20% of cells expressing this construct contained more peripheral structures, but these were judged not to be caveolae by light microscopic methods.

 

Figure 6
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  		Fig. 6. Analysis of caveola formation using deletion mutants and hybrid proteins. (A) Topology model of caveolin. Orange boxes mark the region of interest that are shown in alignments below. (B) Sequence alignment. Dark grey, identical residues; light grey, similar residues; purple, partly conserved hydrophobic residues; red asterisks, residues mutated in breast cancer; shaded yellow region, residues that can be deleted without affecting caveola formation; red line and red text, residues that are not conserved in CeCav-a compared with all other sequences in the alignment; black asterisks, residues implicated in muscular dystrophy when mutated in HsCav3. (C) Caveolin mutants used in this study. Grey, N- and C-terminal regions; dark blue, oligomerisation domain; light blue, scaffolding domain; red, intramembrane domain. Curved lines indicate missing sections. Regions highlighted in green are from CeCav-a. Orange, regions of interest that are shown in alignments. + and – presence and absence, respectively, of caveolae according to the results of the EM caveola biogenesis assay. Scale bars indicate the number of amino acids.

 

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© The Company of Biologists Ltd 2008