1
Caveolin-1 isoform reorganization studied by image correlation spectroscopy

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

2
Caveolae are small, flask shaped invaginations in the cell membrane.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

3
They are thought to play a crucial role in cell signaling, endocytosis and intracellular cholesterol transport.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

4
Caveolin-1, 2 and 3 are key proteins, which are important for the formation of the invaginations on the cell surface.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

5
Caveolin-1 exists in two isoforms: caveolin-1 alpha (α) and caveolin-1 beta (β).

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

6
Little is known about the difference between these two isoforms, and less in known about their role in cell signaling.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

7
Bone morphogenetic proteins (BMPs) are a subfamily of the TGF beta superfamily and their response is mediated by serine/threonine kinase receptors.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

8
Epidermal growth factor (EGF) is known to signal through tyrosine kinase receptors of the ErbB family.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

9
Here we report on the aggregation and association of caveolin-1 isoforms with these receptors and the effect of BMP and EGF activation on caveolin-1 distribution in A431 cells.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

10
Our data, obtained by application of a family of image correlation spectroscopy tools, indicate that BMP and EGF stimulation lead to a rearrangement of the caveolin-1 isoforms on the cell surface.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

11
BMP as well as EGF stimulation leads to a rearrangement of the caveolin-1 β isoform into domains enriched in the caveolin-1 α isoform.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

12
We further show that about 20–30% of the caveolin-1 present at the surface of the cells co-localize with the EGF and BMP receptors.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

13
Using a reporter gene assay sensitive to the activation of the BMP pathway, we show that overexpression of caveolin-1 β inhibits signaling.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

14
Our data suggest that the two isoforms of caveolin-1 play different roles on the cell surface and that caveolae are dynamic structures.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2

Introduction

15
Caveolae are small, flask-shaped invaginations of the cell membrane, which are thought to play a crucial role in endocytosis, signaling and intracellular cholesterol transport.1–3

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

16
Caveolin-1, 2 and 3 are the key proteins involved in the formation of the invaginations.4–6

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

17
Caveolin-1 is expressed in two isoforms, caveolin-1 α and caveolin-1 β, that differ in their coding.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

18
Still, the two caveolin-1 isoforms have a common hydrophobic stretch of amino acids, a scaffolding domain and an acetylated C-terminus.7,8

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

19
They are distributed slightly differently on the cell surface, and previous work suggests that there are caveolae composed only (or mainly) of the β isoform as well as caveolae with both isoforms present.9

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

20
Bone morphogenetic proteins (BMPs) play crucial roles during embryonic development in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis.10–17

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

21
They exert their function by signaling through serine threonine kinase receptors.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

22
BMPs need to bind to at least one type-I and one type-II receptor in order to activate the Smad or p38 pathways.18–28

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

23
Epidermal growth factor (EGF) is in part responsible for the proliferation of cells and exerts its function by signaling through tyrosine kinase receptors, leading to the activation of the Map kinase (Mapk) pathways.29–31

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

24
There is increasing evidence that domains on the cell surface exist to act as sites for protein–protein interactions leading to activation of specific signaling pathways.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

25
By serving as regions of the cell surface that can be enriched in receptors to signaling proteins, the probability of interaction among the receptors increases significantly allowing for more rapid responses.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

26
In the case of the BMP receptors, their organization in caveolae may facilitate creation of preformed complexes of type-I and type II receptors.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

27
Interactions between receptors and regulatory proteins within the domains can, in principle, lead to either activation or inhibition of the corresponding signaling pathways.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

28
However, unless the caveolae are specific to particular receptor families, the high density of receptors in the domain could lead to interaction between different receptor systems and hence cross-talk between otherwise distinct signaling pathways.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

29
It is, therefore, important to study protein–protein interactions within these domains and to determine whether these interactions can change.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

30
Generally, we aim to understand whether stimulation of a cell with receptor specific ligands influences the receptor distribution or the caveolin composition within the caveolae.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa2

31
In this work, we investigate the association of caveolin-1 isoforms with the BMP receptors of type-II (BRII) and type-Ia (BRIa) and with the EGF receptor, ErbB1.

Type: Goal | Advantage: None | Novelty: None | ConceptID: Goa1

32
We apply image correlation spectroscopy techniques to determine quantitatively the density of the receptors and the caveolin-1 isotypes as well as the extent to which they co-localize.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

Materials and methods

Materials

33
Recombinant BMP-2 was obtained from Wyeth (Boston, NY, USA) and human recombinant EGF was purchased from Sigma (St. Louis, MO, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

34
The polyclonal antisera against the BMP receptors, BRIa and BRII, and the polyclonal antibody against the EGF receptor were from Santa Cruz (Santa Cruz, CA, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

35
The antibodies recognizing only caveolin-1 α (clone 2234) or both of the caveolin-1 α and β isoforms (clone 2297) were purchased from Transduction Laboratories (New York, NY, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

36
The secondary antibodies, donkey anti-goat RRX and goat anti-mouse Alexa Fluor 488, were purchased from Molecular Probes (Eugene, OR, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp4

37
The A431 cell line (CRL 1555) was purchased from American Type Culture Collection (Manassa, VA, USA).).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp5

38
The plasmid encoding GFP-tagged caveolin-1 β was provided by Dr H. Kogo (Department of Anatomy, Nagoya University School of Medicine, Showa-ku, Nagoya, 466-8550, Japan.)

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp6

Immunofluorescence labeling of cell surface receptors

39
To measure the distribution of the caveolin-1 on the cell surface, we employed ICS and ICCS measurements, which require acquisition of high quality fluorescence images with a laser scanning confocal microscope.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

40
Normal A431 cells were grown on 22 mm coverslips.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp7

41
Following 72 h of serum-starvation, the A431 cells were stimulated, or not stimulated, with 20 nM BMP-2 for 2.5 h or 100 ng mL−1 EGF for 30 min and fixed using acetone/methanol fixation.32

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

42
After blocking for 30 min with 5% BSA, the cells were incubated with polyclonal antisera recognizing either BRII or BRIa, with polyclonal antibody recognizing the EGFR, or with antibodies recognizing caveolin-1 α (clone 2234) or caveolin-1 α and β (clone 2297) according to manufacturers protocols.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

43
The cells were washed 3 times with PBS for 5 min and afterwards, they were incubated with the corresponding fluorescently tagged secondary antibodies at a concentration of 20 μg mL−1.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp10

44
Cells were washed again three times for 5 min with PBS and the coverslips were mounted on airvol and dried overnight.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp11

Luciferase reporter assay

45
A431 cells grown in 60 mm dishes were co-transfected with the plasmid containing a Smad Binding Element (pSBE), which is sensitive to the activation of the BMP signaling pathway, a Renilla luciferase reporter construct (which is used for normalization of the chemiluminescence response) and the caveolin-1 β construct using Fugene6 (Boehringer).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp12

46
After transfection, the cells were stimulated, or not stimulated, for 12 h with 20 nM BMP-2, lysed and the luciferase activity was measured using a dual luciferase assay system (Promega, Madison, WI, USA).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp13

Confocal microscopy

47
Labeled cells were visualized using a Biorad MRC 600 confocal microscope equipped with an Ar/Kr mixed gas laser and using the appropriate filter sets for dual fluorophore imaging.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

48
Cells expressing the receptors were selected under mercury lamp illumination using a 60 × (1.4 numerical aperture) objective on an inverted Nikon microscope.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

49
An area on the cell, removed from the nucleus, was enlarged and recorded at the zoom 10 setting.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

50
The filter wheel was set for 488 nm laser excitation to measure Alexa Fluor 488 fluorescence, and neutral density filters were used to attenuate the laser to 1% laser power.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

51
Fifteen scans were accumulated on photomultiplier tube 2 (PMT2) in the photon counting mode (to ensure linear scaling of the intensity).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

52
The filter wheel was then shifted to allow excitation with the 568 nm laser line for Alexa Fluor 568 fluorescence, and 20 scans were accumulated on PMT1 for BMP receptors, whereas 9 scans were accumulated on PMT1 for the EGF receptor (EFGR).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

53
Each of the photomultiplier tubes were set to a black level of 6.0 on the vernier scale and a gain setting of 10.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

54
After collection of each set of images was finished, additional images were collected using identical settings but with the shutter to the sample closed to obtain a measure of the dark current for each PMT.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp14

Correlation spectroscopy

55
Image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) are two techniques that can be used to study the distribution and localization of receptors.33–38

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

56
The technique and selected applications have been presented previously at Faraday Discussion .11139

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

57
Here we provide a brief overview of the techniques and their interpretation.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

Image correlation spectroscopy

58
ICS involves autocorrelation analysis of the intensity fluctuations within confocal images collected, in this case, from cells that contain immunofluorescently labeled proteins.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

59
If the fluorescence intensity in a pixel, located at position (x,y) in the image, is i(x,y), then the corresponding normalized fluorescence intensity fluctuation, δi(x,y), is given by: The normalized spatial autocorrelation function, g(ξ,η), is then given by: g(ξ,η) = 〈δi(x,y)δi(x + ξ,y + η)〉where the angular brackets indicate the average over all spatial coordinates and ξ and η are position lag coordinates for the x and y axes in the image.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

60
It is known that the limit of the autocorrelation function as ξ and η approach zero, g(0,0), is equal to the variance of the normalized intensity fluctuations.40

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

61
It is also known that for homogenous, non-interacting species, where the intensity is a true representation of the concentration, the variance of the normalized intensity fluctuations is equal to the variance of the concentration fluctuations, which in turn is equal to the inverse of the number of particles in the observation area, N̄p.41

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

62
In practice, the two-dimensional autocorrelation function for the image is obtained by first calculating the Fourier transform of the image, then calculating the power spectrum (by multiplying the real and the imaginary components of the transform) and finally calculating the reverse Fourier transform.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

63
The resulting two-dimensional autocorrelation function is then fit to a two-dimensional gaussian function of known width (corresponding to the width of the laser beam at the focal point of confocal microscope).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

64
The amplitude of the function obtained from this fit gives the best estimate of g(0,0) for that image.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod2

65
Once the g(0,0) value is known, an important parameter, called the cluster density (CD) can be calculated by eqn. (4).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

66
The CD value is an estimate of the average number of receptor clusters per unit area i.e.,

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod3

Image cross correlation spectroscopy

67
Image cross-correlation spectroscopy (ICCS) is an extension of ICS.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

68
In ICCS, a cross-correlation function is also generated to measure the coincident fluorescence intensity fluctuations in images collected from two different chromophores attached to two different proteins or receptor types.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

69
One receptor type is labeled with a green probe and is imaged to give the intensity map, ig(x,y), while the second receptor type is labeled with a red probe and imaged to give the intensity map, ir(x,y).

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

70
The normalized, spatial cross-correlation function can be calculated as: ggr(ξ,η) = 〈δig(x,y)δir(x +ξ,y + η)〉

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod4

71
In this case, the two-dimensional cross-correlation function is obtained by first calculating the Fourier transform of each of the two images, then cross-multiplying the real part from the green image with the imaginary part from the red image (or the imaginary part from the green image with the real part from the red image, the result is the same) and finally calculating the reverse Fourier transform of the ‘mixed’ power spectrum.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod4

72
The resulting cross-correlation function is fit to a two-dimensional gaussian as above to give the amplitude of the cross correlation function, ggr(0,0).

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod4

73
It has been shown for individual molecules that the zero-lag amplitude of the cross-correlation function reflects the extent of co-localization of the two chromophores on the cell surface.35,36

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

74
We interpret the amplitude to reflect the average number of clusters with both chromophores present.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

75
Specifically: Here, N̄g, N̄r and N̄gr are the average number of clusters which are made up of only green labeled proteins, only red labeled proteins, and both green and red labeled proteins, respectively.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod5

76
Also, gg(0,0) and gr(0,0) are the autocorrelation functions corresponding to the individual green and red images.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod5

77
These are calculated from the individual images as indicated above.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

78
By rearrangement, we can define a cluster density of co-localized chromophores as: The fraction of each protein type associated with the other type can finally be estimated by looking at the appropriate ratios of CD values: In eqn. (8), F(g/r) represents the fraction of green labeled protein clusters which contain red labeled proteins.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

79
Correspondingly, F(r/g) is the fraction of red labeled protein clusters containing green labeled proteins.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod6

80
It can be seen that ICCS provides quantitative information about the extent of co-localization of two different receptors, or in the current work, a receptor and a binding protein.

Type: Method | Advantage: Yes | Novelty: New | ConceptID: Met1

81
The only caveat is that the interpretation of eqn. (6) is strictly only valid for individual chromophores.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod5

Statistics

82
The experiments are designed to collect a large number of images, typically forty, each from a different cell in the sample.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp15

83
Past work indicates that the largest source of uncertainty in the ICS and ICCS measurements arises from variations between cells in a population.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac16

84
In order to obtain a reasonable estimate of the population properties (cluster density, fraction of co-localization) the data are pooled and averaged when more than one experiment is conducted.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

85
In all cases, the cluster density data are represented relative to the value for one, selected control experiment.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

86
All the data in a particular experiment are normalized to this same data set.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

87
The values of the standard error of the mean (SEM) were calculated from the raw data at the 95% confidence level.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

88
The significance of changes relative to the control was assessed by paired, two-tailed t-tests.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

Results

Stimulation of A431 cells causes rearrangement of caveolin-1 isoforms

89
Normally cells are grown in a culture medium to which 10% fetal bovine serum is added.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp17

90
The serum provides some nutrients, but most importantly, a series of growth factors that stimulate cell proliferation.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

91
In the absence of the serum, cells divide less frequently, but grow well for up to one week.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

92
Removal of the serum, serum starvation, is used to isolate the effect of a specific growth factor on cell signaling, but can also affect the receptor concentration on the cell surface by up- or down-regulation at the gene expression level or through altered turn over through endocytosis.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

93
Specifically, serum starvation of A431 cells leads to the up-regulation of the BMP-type-II receptor within 72 h as seen by reverse transcription PCR (RT-PCR) and by immunofluorescence.42

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac17

94
Further, starved A431 cells are sensitive to both BMP-2 and EGF stimulation whereas normal A431 cells are less sensitive to BMP-2 stimulation.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac18

95
A431 cells were starved for 72 h and then stimulated (or not stimulated in control experiments) with the desired signaling protein.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

96
The cells were exposed to BMP-2 for 2.5 h or to 100 ng mL−1 of EGF for 30 min.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp8

97
The cells were then fixed and labeled for the caveolin-1 isoforms using a monoclonal antibody against caveolin-1 α or a monoclonal antibody recognizing both α and β isoforms followed by a secondary fluorescent antibody.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

98
Figs. 1A and 2A show representative confocal images at high magnification (zoom 10 provides 15.5 μm × 15.5 μm images).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs1

99
Forty high magnification images of flat regions of the cell surface were collected from forty separate cells and subjected to ICS analysis to provide a cluster density for each image (eqn. (4)).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

100
The average cluster density was calculated for this large number of images in order to obtain good population statistics.

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

101
Fig. 1B indicates that BMP-2 stimulation of starved A431 cells leads to a 39% decrease in the cluster density of caveolin-1 α and β as detected by labeling with the caveolin-1 antibody clone 2297 (p = 8.75 × 10−5).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs2

102
However, BMP-2 stimulation does not significantly change the cluster density of caveolin-1 α when detected alone by clone 2234 (p = 0.97).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs3

103
This suggests that the change in cluster density of caveolin-1 α and β arises from changes in the distribution of the caveolin-1 β isoform.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res3

104
Most likely, BMP-2 stimulation leads to a redistribution of the caveolin-1 β isoform into regions enriched in the α isoform, since the CD of caveolin-1 α and the CD of caveolin-1 α and β are not significantly different following the BMP-2 stimulation (p = 0.14).

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con3

105
Correspondingly, Fig. 2B shows that EGF stimulation leads to a 37% decrease in the cluster density of the caveolin-1 α and β isoform (detected by clone 2297) (p = 0.013) and that the stimulation with EGF does not significantly alter the cluster density of the caveolin-1 α isoform (detected by clone 2234) (p = 0.96).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

106
This observation is the same as that observed following BMP-2 stimulation.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs4

107
Thus, EGF stimulation leads to a redistribution of the caveolin-1 β isoform into regions enriched in the caveolin-1 α isoform, since the CD of cavolon-1 α and the CD of caveolin-1 α and β are not significantly changed following EGF stimulation (p = 0.84).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res4

108
Evidently, EGF and BMP-2 cause comparable changes in the caveolin isoform redistribution: in both cases the caveolin-1 β isoform is reorganized into domains enriched in caveolin-1 α following exposure to the signaling protein.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

Caveolin-1 isoforms co-localize with BMP and EGF receptors

109
As described above, stimulation of A431 cells with BMP or EGF influences the aggregation of caveolin-1 β and appears to lead to changes in the composition of the caveolae.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

110
Since EGF and BMP mediate their signal through receptors that act respectively as serine threonine kinases or tyrosine kinases, it is likely that the influence on the caveolae compositions are caused by direct interactions with the caveolins, rather than through their respective signaling events.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp1

111
We, therefore, examined the extent of co-localization of the receptors with each of the caveolin-1 isoforms using image cross-correlation spectroscopy (ICCS).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

112
Serum starved A431 cells, stimulated or not stimulated with BMP-2 or EGF, were fixed and the BMP receptors, EGF receptors and caveolin-1 isoforms were labeled using the specific antibodies against the proteins of interest and appropriate secondary fluorescent antibodies.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp9

113
The BMP and EGF receptors were labeled with donkey anti goat Alexa Fluor 568 conjugated secondary antibodies, whereas the caveolin-1 isoforms were labeled using goat anti mouse Alexa Fluor 488 secondary antibodies.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac19

114
Figs. 3A and 4A show representative examples of confocal images of selected, pair-wise labeling of a receptor and a caveolin-1 isoform.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs5

115
It is evident that the extent of co-localization is less than 100% and therefore some of the receptors are outside the caveolae and some of the caveolae contain no receptors.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res5

116
High magnification confocal images were collected and analyzed by ICCS to yield estimates of the density of receptor clusters, the density of caveolin clusters and the density of clusters that contain both receptor and caveolin (eqn. (7)).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

117
The percent of caveolin-1 co-localizing with the BMP or EGF receptors was estimated using eqn. (8).

Type: Method | Advantage: None | Novelty: New | ConceptID: Met1

118
Fig. 3B shows that prior to stimulation with BMP-2 the fraction of co-localization of caveolin-1 α, as detected with clone 2234, and BRIa is about 20% (one fifth of the caveolae contain the receptor) and that after stimulation, the fraction of co-localization is about 34% (one third of the caveolae contain the receptor).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

119
Stimulation by BMP-2 increases the co-localization by about 43% (p = 3.4 × 10−7).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs6

120
In contrast, the co-localzation of caveolin-1 α and β, detected with clone 2297 and BRIa is not significantly affected BMP-2 stimulation remaining at about 26–27% (p = 0.95).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs7

121
The combination of these observations, increase in co-localization with caveolin-1 α and no change with both, indicates that less caveolin-1 β co-localizes with BRIa following stimulation with BMP-2.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

122
The implication is that BMP-2 stimulation may form caveolae enriched in the caveolin-1 α isoform and BRIa.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp2

123
Fig. 3B also shows that BMP-2 stimulation does not significantly change the fraction of co-localization of caveolin-1 α and β with BRII, irrespective of which antibody is used to detect the caveolin-1 (clone 2297 or clone 2234) (p = 0.96 for clone 2234 and p = 0.98 for clone 2297).

Type: Result | Advantage: None | Novelty: None | ConceptID: Res7

124
This indicates that BRIa organization is more flexible than BRII on the cell surface..

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con4

125
Fig. 4B shows that EGF stimulation increases the co-localization of caveolin-1 α with ErbB1 by about 50% (p = 2.93 × 10−5).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs8

126
However, the co-localization of caveolin-1 α and β with ErbB1 is not changed significantly following EGF stimulation (p = 0.62).

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs9

127
This observation is parallel to that observed for BRIa in Fig. 3B and the conclusion is equivalent: Stimulation of A431 cells by EGF causes the redistribution of caveolin-1 β into domains enriched in caveolin-1 α and the increase in caveolin-1 α co-localization with ErbB1 provides evidence for the existence of caveolae enriched in the caveolin-1 α isoform and ErbB1.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

Overexpression of caveolin-1 beta leads to an inhibition of BMP signaling

128
Previous studies have shown that the interaction between the EGF receptor (ErbB1) and caveolin leads to inhibition of the kinase activity of the receptor.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac20

129
Since EGF stimulation causes a redistribution of caveolin-1 β it is possible that it is this isoform that specifically causes the inhibition and that the redistribution is intended to remove the inhibitory effect to allow signaling to take place.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp3

130
Since the BMP-2 and EGF stimulation cause the same effect, the first step is to determine whether there is inhibition of the BMP signaling when the interaction with the caveolin-1 β isoform is enhanced.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp4

131
Accordingly, we transfected the A431 cells with the caveolin-1 β isoform causing an overexpression.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

132
We then used a reporter gene assay to examine the influence of this overexpression on the activation of the Smad signaling pathway.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

133
Specifically, we co-transfected pSBE (with a Renilla luciferase construct) into A431 cells since the pSBE is known to be responsive to the activation of BMP signaling.43

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp16

134
As Fig. 5 shows, BMP-2 stimulation leads to a four-fold increase in luciferase activity in these starved cells.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

135
However, co-expression of caveolin-1 β with the pSBE in A431 cells leads to a decrease in luciferase activity back to the level of unstimulated A431 cells even when they are stimulated with BMP-2.

Type: Observation | Advantage: None | Novelty: None | ConceptID: Obs10

136
This suggests that the presence of excess caveolin-1 β isoform inhibits the BMP-2 signaling pathway.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

137
This is consistent with the previous studies on the ErbB1 that show that interaction of caveolin-1 with the EGF receptor inhibits receptor kinase activity, although in that work it was not attributed specifically to the caveolin-1 β isoform44.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

Discussion

138
Caveolin-1 can exist as two isoforms differing in their N-terminus.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

139
There is evidence that these isoforms play different roles in caveolae formation.9

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

140
Caveolae enriched in caveolin-1 β are shallower than caveolae containing both caveolin-1 α and β.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac21

141
In this work, we show that BMP and EGF receptors co-localize with these caveolin-1 isoforms in starved A431 cells to comparable extents.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

142
Stimulation of these starved A431 cells with either BMP-2 or EGF leads to comparable redistributions of caveolae enriched in the caveolin-1 β isoform into caveolae containing both caveolin-1 α and β isoforms.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

143
We are further able to show that the co-localization of BRIa with caveolae enriched in the caveolin-1 β isoform is decreased upon BMP-2 stimulation.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res6

144
Similarly, we show that stimulation with EGF leads to an increase in the co-localization of caveolin-1 α with ErbB1, whereas the co-localization of caveolin-1 α and β with ErbB1 remains unchanged.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res8

145
The cluster density of caveolin-1 α and β is decreased upon stimulation with BMP-2 or EGF, showing a redistribution of caveolin-1 β into domains enriched in caveolin-1 α.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res9

146
This provided new evidence for the existence of caveolae enriched in the caveolin-1 α isoform only.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con5

147
It is evident that the interaction of the receptors (BRIa and ErbB1) with the caveolin-1 isoforms is sensitive to the state of activation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con6

148
The fact that both BMP-2 and EGF induce the same redistribution of caveolin-1 suggests that the phenomenon may be general and that the redistribution is functionally important in the signaling process.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con7

149
We demonstrate that overexpression of the caveolin β isoform in starved A431 cells leads to an inhibition of BMP-2 induced signaling.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

150
This provides evidence that the caveolin-1 β isoform may inhibit signaling in caveolae enriched in the caveolin-1 β, suggesting that these caveolae may function to sequester the receptors in non-functional domains until they are needed, that is, they may act as storage units for the receptors.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con8

151
Upon stimulation, the receptors are released from the caveolin-1 β inhibitory effect as the caveolin-1 β redistributes.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res10

152
It is known that BMP receptors can form preformed complexes between BRIa and BRII prior to BMP-2 binding, but these preformed complexes do not signal in the absence of BMP-2.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac22

153
Since BRII is a constitutively active kinase there must be a mechanism whereby the transphosphorylation of BRIa by BRII is silenced in the absence of BMP stimulation.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp5

154
It is possible that the caveolin-1 β enriched domains serve this function.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp5

155
In other words, the preformed complexes are inhibited by caveolin-1 β and the inhibition is released as a consequence of the redistribution of the caveolin-1 β in response to the BMP-2.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp5

156
An analogous inhibitory effect may be exerted on the ErbB1 receptor.

Type: Hypothesis | Advantage: None | Novelty: None | ConceptID: Hyp5

157
Whatever the purpose, it is evident that there is an active and dynamic redistribution of caveolin-1 on the cell surface following BMP or EGF stimulation.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con9

158
This demonstrates that caveolae are dynamic structures on the cell surface whose composition may change to influence the signaling activity.

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con2