Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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High resolution three-dimensional imaging of membrane junctional
protein dysfunction using deconvolution microscopy
D. Segretain1,2, J. Gilleron1,§, J. Dompierre2, D. Carette1, J.-P. Denizot3 and G. Pointis1
1
INSERM U 895, University Nice Sophia Antipolis, 151 route Saint-Antoine de Ginestière, BP 2 3194,
06204 Nice cedex 3, France.
2
UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris, France.
3
C.N.R.S., Unité de Neurosciences Information et Complexité, 91198 Gif-sur-Yvette, France
§
Present address: Zerial Laboratory, Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01309, Dresden, Germany.
Epithelial and endothelial barriers are fine organizations of membranous junctions and the integrity of these barriers is
critical to human health. Regulation of membrane junctional proteins depends on a fine-tuned balance resulting from gene
transcription, mRNA processing, protein synthesis, post-translational modifications. Phosphorylation status of junctional
proteins and their associations with other protein partners also play an essential role in this control. This occurs for the
constitutive proteins of gap junctions, connexins (Cx), key regulators of cell proliferation, differentiation and apoptosis,
which exhibit altered trafficking in most tumoral tissues. Conventional light microscopy has been first used to demonstrate
a rapid alteration of Cx trafficking during the first stage of tumoral development. If electron microscopy confirmed such
membranous modifications, it appeared that a new step in optical microscopy was requisite to better understand these
mechanisms. We have then developed a new high-resolution deconvolution microscopy near/under the optical resolution
limit. Such a microscopy approach allowed us to determine the fine three-dimensional (3D) organization of the tissue,
demonstrating that the first step of epithelial tumour progression is a cytoplasmic delocalization of the junctional proteins
from the plasma membrane to the cytoplasm. The major interest of such a system is the possibility to image with highresolution several channels in 3D with fast time acquisition that allowed us to analyze: 1) the molecular events precluding
and participating in endocytosis of Cx; 2) the involvement of Cx partners in these processes; 3) the kinetic of tumoral
agent effects on membranous protein delocalization. In addition, correlative light and electron microscopy (CLEM) have
been successfully developed to determine the fine spatial organization of Cx trafficking. These imaging approaches, which
allow a better understanding of membrane junctional protein dysfunction in disease, could favour the development of new
therapeutic strategies.
Keywords junctional protein, trafficking, deconvolution microscopy, 3D, cancer
1. Introduction
Epithelial and endothelial barriers are fine organizations of several types of junctions (adherens, tight and gap junctions)
that are highly intermingled at these levels. The integrity of these barriers is critical to human health but there is still a
lack of detailed mechanistic knowledge of how the barrier is locally controlled or is able to respond to pathological and
pharmacological effectors. This absence of information limits our understanding of barrier dysfunction in disease and
lowers the development of new therapeutic strategies.
If the overall levels of the proteins that constitute these barriers and their biochemical changes are easy to approach
by means of western blotting analysis, the accurate modifications of their precise localization and trafficking, key
events of disease, in the course of pathological development remain to be investigated.
Our laboratory took a particular interest in connexins (Cxs). Briefly, Cxs are the constitutive proteins of gap
junctions, which allow the intercellular exchange of small signaling molecules (< 1kD) between the cytoplasmic
compartments of two adjacent cells [1]. Cxs are involved in the regulation of cell growth, tissue differentiation,
homeostasis and neoplasic transformation [2]. In many tumor cells, Cxs are expressed but aberrantly localized within
the cytoplasm. This aberrant localization of Cxs has been reported in many human tumoral tissues: primary human liver
tumors, hepatocarcinoma, colorectal carcinogenesis, breast tumors, ovarian carcinoma, endometrial cancer, bladder
cancer, prostate cancer, seminoma and carcinoma-in-situ [3]. Abnormal localization of Cxs has also been reported in the
majority of tumor cell lines studied and in cell lines induced by tumor promoters [4, 5] (see figure 1). By means of
calcein, a fluorophore able to pass between viable cells coupled by gap junctions, we demonstrated that the
mislocalization of Cxs in a mouse Sertoli cell line was preceded by disruption of cell/cell contact and absence of calcein
transfer in DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and HCH (γ-hexachlorocyclohexane) treated cells
[6,7]. As we reported in in vitro models, we also demonstrated that Cxs delocalization and internalization was an early
event of the carcinogenic process by analyzing the kinetic of tumor progression in transgenic mice that develop testis
cancer [8] (see also Fig 1).
Generally, Cxs are distributed in the cytoplasm in both tumor tissues and tumor cell lines, but in almost all studies the
precise intracytoplasmic compartments, the altered steps of Cxs trafficking, the mechanisms and the proteins partners
involved step by step in this defect, have not been identified due to the lack of performing microscopic approaches. Cxs,
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when they are transfected into cells, form large membranous clusters that can be easily detected when these proteins are
tagged with fluorescent proteins or with specific antibodies. Then, the development of performing microscopy image
analysis appears necessary to a better understanding of the fine 3D organization of these structures, their morphological
changes and their interactions with other protein partners essential for their formation and endocytosis and degradation.
The resolution equation of lens-based light microscopy, theorized by Ernst Abbe in 1873 [9], supports the hypothesis
that two objects can not be resolved if they are separated by less than 200nm. Recently, several studies demonstrated
that this limit can be broken through physical (STED) and numerical (PALM/STORM) calculations of the center of
mass of single particles, reaching a resolution close to 20-50 nm [10, 11]. Nevertheless, this super-resolution
microscopy is highly limited in space (3D) and time (live cell imaging) resolution. On the contrary, fluorescence widefield microscopy coupled with deconvolution analysis permits to obtain 3D fast acquisition during time, but with
resolution limited by the Abbe’s equation. At the limit of the resolution spectrum, electron microscopy provides near
atomic resolution but implies fixed thin-sectioned samples, avoiding live-cell imaging. In this domain, highly recent
progresses have been made on both sample processing (high pressure freezing, cryosectioning ...) and acquisitions (3D:
tomography), to allow a more appropriate analysis [12, 13, 14]. Today, it is tempting to consider that the correlation of
the high resolutive electron microscopy with the fluorescence live cell imaging is one of the best approaches to analyze
any biological system. However, such a correlation is not trivial and further studies must be performed to develop the
most appropriate method. The Tokuyasu cryosection method seems to be the most promising way to reach a good
correlation. However, the material needed is not available for all laboratories and not easy to handle. We tried to
develop here a simple method, accessible to almost everybody, which allows us to correlate 3D deconvolution
microscopy with electron microscopy. In order to analyze the alteration process that occurs on gap junctions during
tumoral formation, the correlation of several microscopy tools have to be used. Indeed, if the kinetic of the phenomenon
can be followed by live cell imaging, the high resolution 3D analysis has to be processed through deconvolution
microscopy, and finally the ultrastructural information can only be analyzed by electron microscopy.
2. Technique’s steps
To simultaneously analyze the fast kinetic of junctional proteins endocytosis, the molecular reorganization of their
partners during this process, and the fine ultrastructure of the newly formed degradative vesicular structures, we
developed a high-resolution deconvolution microscopy associated with image treatment by AMIRA software and a
correlative light and electron microscopy method (CLEM). The Figure 2 is a schematic representation of the different
steps that we performed. Briefly, we realized 1) optical sectioning in 2) multiple dimensions (3D, n channels and time)
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3), a 3D reconstruction has been obtained and 4) colocalization analysis or 5) correlation with electron microscopy to
respectively allow unbiased analysis and ultrastructural organization.
2.1. Cell culture preparation
The most important goal for performing correlative light and electron microscopy is the ability to process the samples
for both methods and to analyze the same cell at the two levels of resolution. Several methods have been described in
the past but most of them appeared really complicated to handle and needed sometimes some very sophisticated
facilities [15, 16]. The approach used here is based on new culture dish from Ibidi (Biovalley, Marne la Vallée, France),
having grid into the dish material. So, we cultured and transfected the cells directly into these dishes, which allows the
live cell fluorescence imaging and also to find easily the cell observed in fluorescence after epon embedding through
the grid.
2.2. Deconvolution microscopy system
Figure 3 A is a photography of the microscopy setup implemented in our laboratory. The system presented in this
review was built by the use of a Nikon TE2000-E inverted wide-field epi-fluorescence microscope fully automatized. In
order to obtain a very fast aquisition in multi-dimension, this microscope excited by a halogen lamp, was equipped
with: 1) the faster filter wheel from Prior, 2) a PIFOC objective piezo nanofocusing system (Physik Instrumente) for
fast and reproducible Z-shift of the objective lens, 3) a high temporal resolution CCD cooled camera, Cool Snap HQ2
from Ropper. This 14-bit dynamic range camera allows a high spacial resolution with a sensor size of 1392 x 1040
pixels of 6.45 x 6.45 µm2.
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To obtain high quality images, a 100 X magnification oil lens corrected for the ultraviolet wavelenght from Nikon has
been chosen and specific filters from Chroma allows to select different excitation peaks (360/40; 403/12; 490/20;
540/25 and 635/20) and emission peaks (457/50; 535/40; 617/73; 685/40).
This set up has simultaneously high resolution, wide range dynamic, very good sensitivity and frame range that are
needed for fast multiple dimension acquisitions.
2.3. Optical sectioning and deconvolution
As mentioned before, the 100 X objective lens has been mounted on a PIFOC objective piezo nanofocusing system
(Physik Instrumente) that allows us to realize a fine, fast and precise Z-shift of the objective lens. This system enables
the acquisition of several focal plane sections along the whole specimen. The PICOF was linked, through a firewire
cable, to the personnal computer and was controlled by the acquisition software from Nikon, NIS element.
After acquisition, a tiff stack of all focal plan sections is generated and then processed on the AutoQuant X
deconvolution software from Media Cybernetics. This software calculates a theoretical point spread function (PSF),
based on the information given by the user. Indeed, the PSF is dependent on the magnification (objective lens), the pixel
size and the number of slices from the stack, but also to the wavelengh used, the reflexion index and the numerical
aperture of the objective lens. On the basis of this information, a PSF was generated and the calculated deconvolved
image was compared to the original image for several rounds (iterations). Since we did not observed any significant
differences between the image deconvolved by this method and after beeds-based PSF calculation, the theorical PSF
calculation was routinely used. The deconvolution method used by the software is a 3D restoration method since each
pixel is reattributed to its original point after algorithm calculation and not eliminated as done by 2D neighbor
algorithm. A good example of the power of such deconvolution analysis is shown in the figure 3 B. Indeed, in the raw
data every signal was reattributed to the original fluorochrom by applying a theoritical PSF that leads to the
establishment of unblurred images. Moreover, such technique eliminates the background noise as other deconvolution
software developed.
2.4. Multiple dimension acquisitions
Our system has been built to permit multiple dimension acquisitions. Indeed, with this approach we can acquired Zstack (3D) during several time points (4D) in several X/Y position (5D) and in several channels (6D). As described
above, the z-stack can be precisely acquired via the PIFOC objective piezo nanofocusing system. Then, the NIS
software from Nikon allows restarting the acquisition over time, by generating time points. This time-lapse acquisition
can be very fast (every second) for short-term periods (several minutes), or less fast (every minute) for long-term
periods (several hours/days). In order to preserve the cell in normal culture condition during long-term time-lapse
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imaging, we updated our system with a home-made custom box chamber, covering the whole microscope except the
camera, which needs to be cooled for a better efficiency. This box is linked to “the cube”, from Life Imaging Services,
which controls the temperature inside the chamber. In case of very sensitive cell type (primary culture cell), a CO2 and
humidity controller can be added.
The microscope has been also completed by an X/Y motorized stage, controlled by the acquisition software, which
enables to acquire several positions on the Petri dish. Even if the number of data to be treated and stored highly
increased with these multipositional acquisitions, quantitative analysis could be easily realized. Moreover, several
parameters (control vs treated) could be simultaneously acquired, decreasing the variability due to experimental
conditions (temperature, etc...). Finally, the Chroma filter set present in the Prior filter wheel allows to sequentially
analyze up to four wavelengths (e.g. DAPI, GFP, mCherry, Alexa647). The novelty of such a system is that all these
independent dimensions can be assembled together depending on the need.
2.5. 3D reconstruction and colocalization analysis
Stacks of images can be visualized in several ways. One can realize a maximal intensity projection on one X/Y plan by
using the open acces ImageJ software. Nevertheless, if this method allows a fast visualization, interesting for analyzing
the time-lapse acquisition, the loss of the 3D information is inconsistent with colocalization analysis as well as for
studying the fine events occuring during time (e.g. vesicle fusion).
A second posibility is to use the 3D reconstruction of ImageJ that generates a shadow of the structure in three
dimensions. Neverthless, the limit of this volume view is that 1) any quantification of the volume is not possible; 2) the
3D observation is based on a rotatory movment effect, which prevents videomicroscopy reconstruction.
Finally, the last method for 3D reconstruction that we developed herein, as depicted in figure 3 B, is based on volume
rendering by generation of isosurface after meshing of similar intensity level. This method, based on the AMIRA
software (TGS), is powerful since the visualization was a real 3D structure that could be rotated in all direction and
zoomed in and out. Moreover, the software allows the 3D reconstruction for time-lapse imaging.
In order to analyze the association between two protein partners, we developed several approaches. First, we
analyzed their colocalization by using Plugins from ImageJ (JACoP, etc...) to generate statistical data. Then, we used
the power of the AMIRA software to determine: 1) the precise localization of one protein compared to another by
visualization and section of the 3D images, 2) the distance between each protein to demonstrate that the observed
colocalization region is higher than the resolution limit of the microscope (200 nm).
2.6. Electron microscopy
After visualization in light microscopy, the cells were fixed with glutaraldehyde 2.5% and post-fixed with osmium
tetroxyde 1% reduced by potassium ferrocyanide [17]. After dehydration, cells were embedded in Epon-812 resin. A
small cube in the selected area was realized under the control of the light microscope, allowing to recover the exact
region of interest containing the cells, as defined with the marked grid. Then, a pyramid was trimmed around the cell of
interest visualized by using the light reflexion of the ultramicrotome. Then, serial 70 nm thin sections were cutted and
mounted on 200 mesh copper grid covered by a formvar film. The grids containing the sections were counter-stained by
uranyl acetate and lead citrate as reported [18]. Observation of the serial sections was realized on a Philips CM-10
transmission electron microscope, with a high voltage of 60 kV.
2.7. Correlative Light and Electron Microscopy
In order to correlate both methods, we proposed to segment the AMIRA 3D volume rendering following the 3
orientations (X, Y and Z) to obtain several slices with a thickness close to the 70 nm thin sections prepared for electron
microscopy. Then, a simple comparison of all of these images, with the electron microscopy micrograph, permits to
determine the best orientation and, when necessary, to reajust it (new segmentation following other orientation).
3. Applications using our model
In light of our previous published data, we hypothesized that c-Src association with Cx43 could be a prerequisite for
gap junction plaque internalization [7]. In order to reveal the close molecular interactions that could occur between
Cx43 and c-Src during gap junction plaque endocytic internalization, 42GPA9 Sertoli cells were transiently transfected
with Cx43-GFP. We first evaluated the number of contiguous cells capable of forming Cx43-GFP plaques and
expressing the tagged Cx43 in both adjacent cells and we evaluated the presence of immunoreactive c-Src (IR c-Src)
near the gap junction plaque with a specific c-Src antibody. Deconvolution analysis of immunofluorescence for Cx43GFP (green fluorescence) and c-Src (red fluorescence) revealed that c-Src was randomly distributed near to the gap
junction plaques but not in direct contact (Fig. 4 A). IR c-Src was detected as small dots on one side of the plaque at the
beginning of the internalization process observed in the presence of the carcinogen HCH (Fig. 4 C). The protooncogene signal was found thereafter in the outer face of the invaginated plaque (Fig. 4 E). After internalization, the
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plaque formed large round structures close to the plasma membrane with a size comprise (0.5-2 µm) identified as
annular gap junction (fig 4 G). Deconvolution analysis of one annular gap junction showed that immuoreactive c-Src
spots were only detected around this structure (Fig. 4 G). Such specific locations of c-Src versus Cx43, during the
sequential steps of gap junction plaque internalization, were confirmed by AMIRA reconstructions of the respective
images (Fig 4 B, D, F, H) that show a specific association between c-Src and Cx43. Tilting angles of a gap junction
plaque (-60°, 0°,+60°, middle panels) or of an annular gap junction (-45°, 0°,+45°, lower panels) reconstructed with
Amira software demonstrate strict association between the kinase and Cx43 (see insets).
In order to examine in every detail the altered membranous cytoplasmic structures and the ultrastructure of the newly
formed annular gap junctions, around which we observed numerous molecular reorganizations, we developed a
correlation analysis between 3D deconvolution microscopy and electron microscopy into Sertoli cells expressing Cx43GFP (Fig 5).
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Transfected cells have been cultured first on small Petri dishes allowing to examine them with the inverted
microscope. The plastic support was engraved forming small squares. Each of them contained a number or a letter.
Thus, it was easy to locate the cell of interest, previously observed and photographed at different magnifications at the
light microscope level. The cells were then fixed and processed as usual for electron microscopy. In the present case,
we focused on a large annular gap junction (Fig 5A left panel). This endocytic structure could be visualized in different
Z sections (Fig 5A right upper panel) and thus a 3D reconstruction in ImageJ software has been performed. This
reconstruction could permit to give a good idea of the structure but could not allow to detect a real correlation with
images from electron microscopy. To do so, we decided to realize 3D volume rendering through the powerful AMIRA
software (Fig 5B). A rotation of 90° of the reconstruction in XY and XZ directions gives a good idea of the spatial
organization of the annular gap junction but also allows us to choose the rotation for the correlation with the electron
microscope image (Fig 5B upper panels). Then, a slicing of the structure was realized to obtain small slices
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corresponding to the 70 nm sections done during the electron microscopy protocol (Fig 5B lower left panel). The slice
from AMIRA (light microscopy) was then merged with the electron microscopy image (Fig 5B lower middle panel) to
generate the correlative image (Fig 5B lower right panel). This technology opens new fields of the fine organization of
the annular gap junction. Indeed, these data clearly demonstrate that the annular gap junction newly formed is
composed by double membranous structure as previously described [19]. It appears here that the arrest or hole present
in the annular gap junction as observed in fluorescence by Amira reconstruction are closed to the plasma membrane,
demonstrating a clusterization of Cx43 protein in specific micro domains of the annular gap junction.
4. Generalization of the methodological approach
Membrane remodelling is the result of a series of molecular interactions more or less sequentially organized. If
molecular and biochemical approaches have allowed to determine some of these interactions, it was necessary to
examine such a complex in situ to visualize the fine molecular alterations associated with pathological situations. By
developing a high resolution deconvolution microscope coupled with 3D Amira reconstructions, it was possible to
examine molecular reorganization near the resolution limits of the microscope. The methodology developed here allows
the fine analysis of molecular remodelling during an active process in two different levels of resolution. If this
methodology was very helpful in our research project, it can be applied in numerous biological contexts in which
videomicroscopy, multiple proteins interactions and ultrastructural analysis are needed. Moreover, the strength of this
methodology is the fact that almost everybody can apply it since its application and processing are easy, of low cost and
not highly specialized.
In term of biological relevance, it is obvious that a method correlating several microscopy approaches are very
helpful. Since the biochemical analysis can give great idea of the protein that interacts but no spatial and temporal
information about this event, it was of major interest to develop such a technology. Moreover, even if we described here
only the cell biological aspect of the results obtained by our system, similar 3D analysis have already be done in ex vivo
tissue [20]. Indeed, we demonstrated a modification of Cx43 localization and expression after several short-time
exposures to testicular hormones. These aspects demonstrate that this methodology can also allow a fine analysis of
proteins reorganization into more complexes but also physiological models. Such an approach could then be used for
analysis of tissue polarization, growth and regulation during development of several organ and/or organism. The fine
ultrastructural analysis can then allow some new insight about the environment of the proteins of interest and why not
some unexpected observations leading to new discovery.
5. Conclusion
There is now clear evidence that several Cx protein partners participate in gap junction endocytosis, but the respective
role and the time at which they are involved are still unknown. The development of innovative and highly resolutive “in
space and time” microscopy is of major interest in order to determine the fine molecular remodelling that precludes and
participates in these processes. By using deconvolution microscopy, we first described the kinetic of gap junction
endocytosis and annular gap junction formation under exposure to a carcinogen. Then, we used the power of the 3D
AMIRA volume rendering to analyze the molecular reorganization of c-Src, ZO-1 and Cx43 during gap junction
internalization [7]. Indeed, we reported that the proto-oncogene c-Src only interacts with Cx43 on one side of the gap
junction plaque. The evidence of this fine molecular organization would not be possible without 3D reconstruction and
rotation of the cellular structures of interest. This innovative 3D methodology could be applied to analyze other Cx
partners such as actin, dynamin or β-catenin. This fine analysis could also allow to determine further the molecular
events that drive membrane junctional protein internalization and their involvement in the tumoral process.
In addition we have associated time-lapse imaging with deconvolution microscopy. This approach allows faster
acquisition than confocal microscopy does and the reduction of the photobleaching favours acquisition for longer times.
Thus, by using this methodology we were able to analyze both relatively slow events for a long period of time, such as
the gap junction plaque endocytosis kinetic, and also very fast events such as vesicles trafficking [21]. Another
advantage of our system is the automatized XY stages that allow multipositioning acquisitions. Indeed, highly
qualitative analysis performed could be coupled with quantitative analysis and generates statistics.
Lastly, we developed a correlative light-electron microscopy method that allows videomicroscopy analysis and
ultrastructural observation of the same cellular structure. Nevertheless, this approach was unlikely for ultrastructural
localization of several proteins. Recently, an innovating optical super resolution microscopy, which bypassed the
resolution limit of fluorescence microscopy, has been developed. By using new probes (photoactivable GFP,
photoactivable TagRFP) and highly sensitive detector (EMCCD camera), localization of single particles can be realized.
This single particle localization allows algorithms to calculte the barycenter of each fluorochrome, leading to a
resolution close to 20 nm in 2D [20] and 3D [22]. Based on our methodology and the super-resolution microscopy, it is
likely to imagine a very efficient technology that could correlate 3D time-lapse GFP imaging, 3D super-resolution
microscopy analysis of several proteins of interest, by using antibodies [23], associated with electron microscopy. Such
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an approach could open new gap for a better knowledge of molecular and ultrastructural molecular reorganization
during physiological and pathological processes.
Acknowledgements Authors are grateful to F. Carpentier for technical assistance and L. Gilleron for reading the manuscript. J.G.
was funded by the French Ministry of Research and Technology, and J. Dompierre by the French Minister of Environment. D Carette
was a fellowship from ANR (05-PCOD-006-02) and from the Association pour la Recherche sur le Cancer (ARC).
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