Lipid-rafts remain stable even after ionizing radiation induced disintegration of β1 integrin containing focal adhesions
Babel et al. BMC Res Notes
Lipid-rafts remain stable even after ionizing radiation induced disintegration of β1 integrin containing focal adhesions
Laura Babel 0 1 2
Larissa Kruse 0 2
Steven Bump 0 2
Markus Langhans 0 2
Tobias Meckel 0 1 2
0 Membrane Dynamics, Department of Biology, Technische Universität Darmstadt , Schnittspahnstrasse 3, 64287 Darmstadt , Germany
1 GRK 1657, Molecular and Cellular Responses to Ionizing Radiation, Technische Universität Darmstadt , Darmstadt , Germany
2 Membrane Dynamics, Department of Biology, Technische Universität Darmstadt , Schnittspahnstrasse 3, 64287 Darmstadt , Germany
Objective: Adhesion of cells to the extracellular matrix is facilitated by integrin receptors. We recently found that a nanoscale organization of plasma membrane located integrins containing the β1 subunit is responsible for an enhanced radio-resistance in 3D cultured cells over cells grown in 2D. While ionizing radiation is known to have broad effects on the lipid composition of the plasma membrane and their organization in lipid-rafts, it is not clear whether the effects of ionizing radiation on the nanoscale clustering of integrins is lipid-raft dependent. Results: Using single molecule microscopy we can show that β1 integrins colocalize with cholesterol in lipid-rafts. Ionizing radiation, as an extrinsic stressor, causes the separation of β1 integrins from cholesterol lipid raft suggesting that the effects of ionizing radiation on the clustering of β1 integrins are lipid-raft independent.
Lipid raft; Membrane dynamics; Integrin; 3D cell culture; Single molecule microscopy
It has been reported that cells embedded in a 3D matrix
are more radio-resistant than those cultured in a
standard, monolayer 2D cell culture. This phenomenon of
increased radioresistance in a 3D matrix has been termed
]. We recently found that clustering of integrin β1
is a sensitive and robust indicator of radio-resistance
]. Cells cultured under standard (2D) conditions are
not able to organize integrin receptors, which facilitate
cell adhesion [
], into firm and stable clusters. They
display a rather loose and dynamic cluster organization of
the ECM (extracellular matrix) receptor. On the
contrary, cells embedded in an ECM, exhibit a stable integrin
organization. Exposure of 2D cultured cells to ionizing
radiation causes already at low doses a severe disturbance
of the unstable integrin organization. The same treatment
has no perceivable effect on the well clustered
organization of integrins in 3D cultured cells. On the basis of
these data we could therefore causally link the
radioresistance of 3D cells to their ability to maintain stable
It is well accepted that IR has profound effects on the
PM beyond integrin clustering. Mainly lipid
peroxidation, generation of ceramides and its organization in
ceramide lipid rafts are well studied. Ionizing
irradiation generates reactive oxygens (ROS) which damage
the integrity of the membrane and modify lipids directly
with the consequence of profound effects on lipid
signalling, organization and dynamics [
differences in lipids such as chain length, chain geometry
and head groups cause an in-homogeneous distribution
of membrane components and an aggregation in defined
domains. In particular sphingolipids and cholesterol
aggregate in microdomains known as lipid rafts [
Lipid rafts are highly dynamic structures, of 10–200 nm
size, which limit the free diffusive properties of
biomembranes as proposed by Singer and Nicolson in their fluid
mosaic model . These micro structures are known to
function as parts of signaling cascades or as platforms for
membrane protein clustering; in this way they modify
protein activity [
]. Proteins localize in lipid rafts either
because of direct interaction with the lipid head group
or in response to physical forces such as lateral pressure,
charge interactions or the local curvature of the
]. It is known that integrins and cholesterol rich
regions colocalize [
] suggesting that integrins are
predominantly localized in lipid rafts.
Here we use ionizing radiation as a tool to disrupt
integrin clustering and native co-cluster organization of
integrin β1 with cholesterol. In the case that lipid rafts
are responsible for the effects on integrin clustering, we
expect that: (i) the before mentioned cholesterol raft
organization is ECM dependent, and (ii) that IR breaks
cholesterol raft organization in concert with integrin
cluster break down.
To our surprise, we found that integrins disintegrate
in a lipid raft independent manner. Even after high doses
of IR cholesterol remained in clusters, while β1 integrins
were separated from their raft localization.
A detailed description of the methods, with references to
], can be found in Additional file 1.
Membrane mobility and lipid raft organization are strongly
affected by the cell culture condition
To investigate the mobility and nanoscale organization of
the PM of cells as a function of their culture conditions,
we analyzed an isoprenyl anchored membrane protein
(CAAX-mCherry) as a reporter for membrane fluidity
] and clustering of cholesterol as a marker for lipid
rafts in 2D and 3D cultured cells.
For the analysis of membrane mobility, cells were
transfected with CAAX-mCherry and the mobility of this
protein was monitored by FRAP (fluorescence recovery after
photobleaching). The recovery curves reveal (Fig. 1a)
that 3D cultured cells possess a higher membrane
fluidity; fluorescence recovery occurred faster than in 2D
cultured cells. An exponential fit yields a halftime recovery
value of 10.63 s and a mobile fraction of 88% for 3D cells.
Corresponding analysis on the top membrane of 2D
cultured cells reveal a similar value for the mobile fraction of
83% but a much longer halftime recovery (27.41 s). These
results show that already the basic fluidity of the PM
differs between 2D and 3D cultured cells. Since basically all
signaling cascades relay on a dynamic (re)organisation
of the PM [
], we can assume that the dynamics of PM
located signaling are bound to differ in 2D and 3D
To further investigate if lipid rafts, often attributed as
the organizers of PM located signaling activity [
affected by the different culture conditions, 2D and 3D
cultured cells were stained with a cholesterol affine
fluorescent probe (Dronpa-θD4). Cells were than imaged by
single molecule localization microscopy and
quantitatively assessed by a detailed cluster analysis (Fig. 1b–g).
Because it was unfortunately not possible to completely
immobilize lipids via chemical fixation [
assured that the remaining mobility was not altering
the cluster organization (Additional file 1: Figure S1).
The effects of the two cell culture conditions on
cholesterol raft organization can be directly recognized by
a visual inspection of the single molecule localization
irng ax 400
isu 400 ****
3D rrad nm200
0 20 40 60Tim8e0/ s100 120 130 ltseuC in 0 2D 3D
Fig. 1 2D vs. 3D cell culture conditions have a strong impact on the membrane mobility and cholesterol raft organization. a FRAP curves of PM
located CAAX-mCherry of 2D (blue, n = 8) and 3D (green, n = 9) cultured OV-MZ-6 cells. Exponential fits of recovery dynamics and the standard
derivations. b–g Single molecule data of cholesterol stainings of 2D and 3D MEF cells as well as corresponding cluster analysis. b, e Scatter plots
show all detected cholesterol molecules, c, f corresponding heat maps visualize clustered (yellow) and unclustered (dark blue) regions, arrows
indicate cholesterol rafts. Scale bar is 1 μm. Statistical analysis with the Ripley’s K function reveals the clustering (d) and the cluster size (g). Statistical
analysis was performed with a Mann–Whitney test. **p ≤ 0.01 and ****p ≤ 0.0001
results. Each point in the scatter plot of Fig. 1b, e
represents an individual detection of a cholesterol molecule.
Both scatter plots show that cholesterol is organized in
micro-domains; this is evident from a higher density of
the signals. These domains, long know as
sphingolipidcholesterol lipid rafts [
] vanish upon cholesterol
depletion (Additional file 1: Figure S2). To quantify the visual
impression we performed a Ripley’s K function cluster
analysis. This function counts the number of signals that
fall within a defined radius of each detected signal. By
plotting this number versus the respective radii a
distribution (H-plot) is yielded. The first local maximum in this
plot represents the most prominent cluster formation of
the data set. The height of this maximum provides: (i) a
measure of the clustering (H(r) max) and (ii) the position
the cluster radius (r max). For a better visualization of the
single molecule localizations, 2D plots of the H(r) max
values are represented as heatmaps. They identify clustered
regions with a higher density of signals as yellow areas
(Fig. 1c, f ). The heat maps reveal that 2D cultured cells
possess more cholesterol rafts with a higher degree of
clustering. The quantitative K function analysis support
these findings (Fig. 1d, g). 2D cultured cells exhibit a
significantly (**p ≤ 0.01) higher degree in clustering
compared to 3D cultured cells. The former also have a smaller
radius (****p ≤ 0.0001: 2D 〜 100 nm, 3D 〜 160 nm).
Taken together the data show that not only the
membrane mobility but also the organization of lipids into
rafts are remarkably affected by the cell culture
condition. This suggests even more that PM located signaling
activity differs in 2D and 3D cultured cells. The results of
these experiments are well in line with our previous
findings in that not only integrin β1 clustering, but also the
number of the immediate downstream signaling partner
pFAK (phosphorylated focal adhesion kinase) differs
significantly between the cultured conditions. 2D cultured
cells presumably possess an impaired signalling
]. At this point we can conclude that the
localization and organization of cholesterol rafts differ in cells
depending on whether they were cultured in 2D or 3D.
Lipid rafts—other than integrins—do not change their
cluster organization in response to high dose irradiation
To examine whether the colocalization of integrin β1
and cholesterol is maintained after high dose irradiation,
we stained cells in order to monitor both micro
organizations. After co-staining the target domains cells were
irradiated and imaged, followed by single molecule
localization analysis. The data reveal a culture condition
independent coclustering of cholesterol rafts and integrin β1
clusters (Fig. 2a, i).
Previously we found that 2D cultured cells have a
less well organized status of integrin β1. These unstable
clusters were easily disturbed even by low doses (2 Gy) of
radiation. In contrast, the same IR dose turned out to be
completely ineffective in 3D cultured cells for affecting
the well clustered organization of integrins. Also a high
dose of irradiation (15 Gy) leads in 2D cultured cells to a
complete break down of integrin clusters while it causes
only a partial disintegration in 3D cultured cells [
If IR induced integrin cluster break-down would mainly
be determined by lipid rafts one would expect that the
same treatment causes a simultaneous disintegration of
both domains. 2D cultured cells, which were fixed 15 min
after an irradiation with 15 Gy, exhibited a loss of
integrin clusters and a decreased amount of integrins. The
cholesterol raft organization on the other hand remained
unaffected by this treatment (Fig. 2b). The results of these
experiments show that the integrin cluster break-down is
unrelated to the integrity of lipid rafts. Heat maps
support this finding (Fig. 2c–h). While the clustering of
cholesterol remains unchanged, integrin clusters and signals
are lost 15 min after irradiation; they only partly
regenerated after 6 h.
In contrast to 2D cells, 3D cells not only maintain their
clustered organization of β1 integrins after irradiation
with high doses but also show a faster recovery.
Irradiation with 15 Gy only triggers a slight decrease in
integrin clustering and therefore also only a minor reduction
of integrin-cholesterol coclustering (Fig. 2j–p) 15 min
after IR. The effects are completely recovered after 6 h.
As much as cholesterol rafts are not affected by high
dose irradiation with 15 Gy in 2D cultured cells they also
remain unaffected in 3D cultured cells. Following visual
inspection of the images we used the Ripley’sK
function to generate H-plots for quantification (Fig. 3). The
H-plots reveal that the cholesterol organization is
unaffected by high dose irradiation in a cell culture
independent manner. Our detailed cluster analysis reveals that also
parameters, such as cholesterol raft density and number
of cholesterol microdomains do not change after
irradiation (Additional file 1: Figure S3). These results
demonstrate, that it is possible to separate a protein from its
lipid raft localization by physical force like X-ray
irradiation. This implies that independent forces underlie
the co-organization of proteins and lipids in membrane
Effects of IR on integrin β1 clustering are lipid raft
Taken together, we found that:
• Membrane dynamics and cholesterol raft
tion differ between 2D and 3D cultured cells.
• The integrin-cholesterol raft colocalization is cell
0 200 400 600 0 200 400 600
r in nm r in nm
Fig. 3 Effects of ionizing radiation on integrin β1 and cholesterol microdomain organization of 2D and 3D cultured MEF cells. H-Plots of
datasets analyzed with Ripley’s K function for integrin β1 and cholesterol microdomains from 2D (a) and 3D (b) cultured cells. The peak heights
(H(r) = L(r) − r) represent the degree of clustering (H(r) max) and their position the most frequent cluster size (r in nm). H-plots show results for
controls and cells irradiated with 15 Gy fixed 15 min and 6 h after IR. Color code: integrin β1 control (black), integrin β1 15 min after IR (dark gray),
integrin β1 6 h after IR (light gray), 2D cholesterol control (dark blue), 2D cholesterol 15 min after IR (mid-blue), 2D cholesterol 6 h after IR (light
blue), 3D cholesterol control (dark green), 3D cholesterol 15 min after IR (mid-green) and 3D cholesterol 6 h after IR (light green). Also, an analysis of
100 random distributions of localizations containing the same number of signals as the control are plotted (confidence interval, gray)
• Integrins can be separated from their lipid raft
zation by an extracellular stressor.
• Cholesterol rafts remain surprisingly stable even
after a sudden and complete disappearance of
proteins, with which they colocalized before a treatment.
Even after exposing cells to high doses of IR, cholesterol
remains clustered in the PM. In contrast, integrin
clusters disintegrate in response to this treatment and loose
their association to lipid rafts, often referred to as
“organizing platforms” [
]. With these experiments we could
show that the effects of IR on the integrin β1 clustering
are lipid raft independent. But our results also pose the
question: who organizes whom? This is a well known
question which is addressed for years in the filed of
Our data indicates that this question has to be
answered with “neither is responsible for the
organization of the other”. While integrins and cholesterol rafts
clearly colocalize under unstressed conditions,
treatment with IR showed that lipid rafts cannot be made
responsible for the clustered organization of integrins.
In other words, cholesterol does not pattern integrins.
On the other hand, the distribution of integrins turned
out not to be responsible for the presence of cholesterol
rafts, as disintegration of the former did not effect the
latter. Hence, patterning processes behind cholesterol and
integrins appear to be independent or at least lack strong
In conclusion, the generalized view of lipid rafts as an
“organizing platform” is questioned by our data at least
for integrins. In this respect our findings are also not in
line with the general view that integrin-signalling
stabilizes lipid rafts [
], as they remained stable in the
absence of intact focal adhesions.
The present data do not provide a complete answer to the
question on “Who organizes whom?”. Our results only
imply that the generalized view of lipid rafts as
organizing platforms has exceptions and needs further review.
Additional file 1. Additional information. Details of experimental
procedure and Additional figures S1, S2 and S3.
ECM: extracellular matrix; SMD: single molecule detection; PM: plasma
membrane; IR: ionizing radiation; CAM-RR: cell-adhesion-mediated-radio-resistance;
FRAP: fluorescence recovery after photobleaching; PALM: photoactivated
localization microscopy; CLSM: confocal laser scanning microscopy; FAK: focal
Conception and design of this work was done by LB. Data collection, analysis
and interpretation was done by LB, SB and LK. LB and TM drafted the
manuscript. Critical revision of the article was done by LB, ML and TM. All authors
read and approved the final manuscript.
We thank Dr. A. Miyawaki (RIKEN Brain Science Institute, Japan) for kindly
providing the pET28/Dronpa-θ-D4 plasmid. We also acknowledge support by
the German Research Foundation and the Open Access Publishing Fund of
Technische Universität Darmstadt.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets generated and/or analysed during the current study are available
from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This work was supported by the Deutsche Forschungsgemeinschaft (DFG,
German Research Foundation) with grants to T. Meckel (Me3712/1-2 and GRK
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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