E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity.

Spheroids resemble features of tissues and serve as model systems to study cell-cell and cell-ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue integrity. Tissue integrity involves the cellular processes adhesion formation, adhesion reinforcement, rearrangement as well as proliferation. They maintain the structure and function of tissues and, upon dysregulation, contribute to malignancy. We investigated spheroid formation dynamics in cell lines of different metastatic potential. We dissected spheroid formation into phases of aggregation, compaction and growth to identify the respective contributions of E-cadherin, actin, microtubules and FAK. E-cadherin, actin and microtubules drive the first two phases. Microtubules and FAK are involved in the proliferation phase. FAK activity correlates with the metastatic potential of the cells. A robust computational model based on a very large number of experiments reveals the temporal resolution of cell adhesion. Our results provide novel hypotheses to unveil the general mechanisms that contribute to tissue integrity.

Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy.

Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verified in toto immunostaining protocols for large multicellular spheroids or for tissue sections have not been published. We present a method for the verification of immunostaining in three-dimensional spheroids. The analysis relies on three criteria to evaluate the immunostaining quality: quality of the antibody stain specificity, signal intensity achieved by the staining procedure and the correlation of the signal intensity with that of a homogeneously dispersed fluorescent dye. We optimized and investigated variations of five immunostaining protocols for three-dimensional cell biology. Our method is an important contribution to three-dimensional cell biology and the histology of tissues since it allows to evaluate the efficiency of immunostaining protocols for large three-dimensional specimens, and to study the distribution of protein expression and cell types within spheroids and spheroid-specific morphological structures without the need of physical sectioning.

Identifying the necrotic zone boundary in tumour spheroids with pair-correlation functions.

Automatic identification of the necrotic zone boundary is important in the assessment of treatments on in vitro tumour spheroids. This has been difficult especially when the difference in cell density between the necrotic and viable zones of a tumour spheroid is small. To help overcome this problem, we developed novel one-dimensional pair-correlation functions (PCFs) to provide quantitative estimates of the radial distance of the necrotic zone boundary from the centre of a tumour spheroid. We validate our approach on synthetic tumour spheroids in which the position of the necrotic zone boundary is known a priori It is then applied to nine real tumour spheroids imaged with light sheet-based fluorescence microscopy. PCF estimates of the necrotic zone boundary are compared with those of a human expert and an existing standard computational method.

Robust and automated three-dimensional segmentation of densely packed cell nuclei in different biological specimens with Lines-of-Sight decomposition.

BACKGROUND: Due to the large amount of data produced by advanced microscopy, automated image analysis is crucial in modern biology. Most applications require reliable cell nuclei segmentation. However, in many biological specimens cell nuclei are densely packed and appear to touch one another in the images. Therefore, a major difficulty of three-dimensional cell nuclei segmentation is the decomposition of cell nuclei that apparently touch each other. Current methods are highly adapted to a certain biological specimen or a specific microscope. They do not ensure similarly accurate segmentation performance, i.e. their robustness for different datasets is not guaranteed. Hence, these methods require elaborate adjustments to each dataset. RESULTS: We present an advanced three-dimensional cell nuclei segmentation algorithm that is accurate and robust. Our approach combines local adaptive pre-processing with decomposition based on Lines-of-Sight (LoS) to separate apparently touching cell nuclei into approximately convex parts. We demonstrate the superior performance of our algorithm using data from different specimens recorded with different microscopes. The three-dimensional images were recorded with confocal and light sheet-based fluorescence microscopes. The specimens are an early mouse embryo and two different cellular spheroids. We compared the segmentation accuracy of our algorithm with ground truth data for the test images and results from state-of-the-art methods. The analysis shows that our method is accurate throughout all test datasets (mean F-measure: 91%) whereas the other methods each failed for at least one dataset (F-measure≤69%). Furthermore, nuclei volume measurements are improved for LoS decomposition. The state-of-the-art methods required laborious adjustments of parameter values to achieve these results. Our LoS algorithm did not require parameter value adjustments. The accurate performance was achieved with one fixed set of parameter values. CONCLUSION: We developed a novel and fully automated three-dimensional cell nuclei segmentation method incorporating LoS decomposition. LoS are easily accessible features that ensure correct splitting of apparently touching cell nuclei independent of their shape, size or intensity. Our method showed superior performance compared to state-of-the-art methods, performing accurately for a variety of test images. Hence, our LoS approach can be readily applied to quantitative evaluation in drug testing, developmental and cell biology.

Lateral assembly of N-cadherin drives tissue integrity by stabilizing adherens junctions.

Cadherin interactions ensure the correct registry and anchorage of cells during tissue formation. Along the plasma membrane, cadherins form inter-junctional lattices via cis- and trans-dimerization. While structural studies have provided models for cadherin interactions, the molecular nature of cadherin binding in vivo remains unexplored. We undertook a multi-disciplinary approach combining live cell imaging of three-dimensional cell assemblies (spheroids) with a computational model to study the dynamics of N-cadherin interactions. Using a loss-of-function strategy, we demonstrate that each N-cadherin interface plays a distinct role in spheroid formation. We found that cis-dimerization is not a prerequisite for trans-interactions, but rather modulates trans-interfaces to ensure tissue stability. Using a model of N-cadherin junction dynamics, we show that the absence of cis-interactions results in low junction stability and loss of tissue integrity. By quantifying the binding and unbinding dynamics of the N-cadherin binding interfaces, we determined that mutating either interface results in a 10-fold increase in the dissociation constant. These findings provide new quantitative information on the steps driving cadherin intercellular adhesion and demonstrate the role of cis-interactions in junction stability.

Basic building units and properties of a fluorescence single plane illumination microscope.

The critical issue of all fluorescence microscopes is the efficient use of the fluorophores, i.e., to detect as many photons from the excited fluorophores as possible, as well as to excite only the fluorophores that are in focus. This issue is addressed in EMBL's implementation of a light sheet based microscope [single plane illumination microscope (SPIM)], which illuminates only the fluorophores in the focal plane of the detection objective lens. The light sheet is a beam that is collimated in one and focused in the other direction. Since no fluorophores are excited outside the detectors' focal plane, the method also provides intrinsic optical sectioning. The total number of observable time points can be improved by several orders of magnitude when compared to a confocal fluorescence microscope. The actual improvement factor depends on the number of planes acquired and required to achieve a certain signal to noise ratio. A SPIM consists of five basic units, which address (1) light detection, (2) illumination of the specimen, (3) generation of an appropriate beam of light, (4) translation and rotation of the specimen, and finally (5) control of different mechanical and electronic parts, data collection, and postprocessing of the data. We first describe the basic building units of EMBL's SPIM and its most relevant properties. We then cover the basic principles underlying this instrument and its unique properties such as the efficient usage of the fluorophores, the reduced photo toxic effects, the true optical sectioning capability, and the excellent axial resolution. We also discuss how an isotropic resolution can be achieved. The optical setup, the control hardware, and the control scheme are explained in detail. We also describe some less obvious refinements of the basic setup that result in an improved performance. The properties of the instrument are demonstrated by images of biological samples that were imaged with one of EMBL's SPIMs.

Three-dimensional optical manipulation using four collimated intersecting laser beams.

The optical Earnshaw theorem states that a small particle cannot be trapped solely by scattering forces. This limitation is overcome in a novel differential all-optical manipulator. It utilizes four collimated laser beams arranged along the axes of a tetrahedron to confine and move a microscopic sample in an aqueous medium. By adjusting the intensity of each beam individually the magnitude and direction of the optical forces acting on the sample, and via these its position, are controlled. Since only scattering forces are exploited the system is not confined to trapping near a geometrical focus, and therefore enables three-dimensional manipulation over ultra-long working distances. Latex beads 20 microm in diameter can be positioned arbitrarily within a volume defined by the overlap of the four 100 microm diameter beams. The sample is observed from four directions simultaneously, demonstrating the instrument's potential as a universal manipulator in connection with high- and isotropic-resolution light microscopy.

Spatial partitioning of secretory cargo from Golgi resident proteins in live cells.

BACKGROUND: To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known. RESULTS: We observed partitioning of the fluorescent Golgi resident T2-CFP and fluorescent cargo proteins VSVG3-YFP or VSVG3-SP-YFP upon Golgi exit after a synchronous pulse of cargo was released from the ER. Golgi elements remained stable in overall size, shape and relative position as cargo emptied. Cargo segregated from resident rapidly by blebbing into micron-sized domains that contained little or no detectable resident protein and that appeared to be continuous with the parent Golgi element. Post-Golgi transport carriers (TCs) exited repeatedly from these domains. Alternatively, entire cargo domains exited Golgi elements, forming large TCs that fused directly with the plasma membrane. However, domain formation did not appear to be an absolute prerequisite for TC exit, since TCs also exited directly from Golgi elements in the absence of large domains. Quantitative cargo-specific photobleaching experiments revealed transfer of cargo between Golgi regions, but no discrete intra-Golgi TCs were observed. CONCLUSIONS: Our results establish domain formation via rapid lateral partitioning as a general cellular strategy for segregating different transmembrane proteins along the secretory pathway and provide a framework for consideration of molecular mechanisms of secretory transport.

Spindle positioning during the asymmetric first cell division of Caenorhabditis elegans embryos.

Cell division during development in many cases generates daughter cells that differ not only in fate, but also in size. We investigate the mechanisms that ensure proper spindle positioning during such asymmetric divisions using the one-cell stage Caenorhabditis elegans embryo as a model system. We utilized a UV laser microbeam as an in vivo microtubule-severing device to probe the forces driving spindle positioning. Our results indicate that extra-spindle pulling forces acting on the spindle poles dictate spindle position along the anterior-posterior embryonic axis. Importantly, forces acting on the posterior spindle pole appear more extensive than those acting on the anterior one, thus explaining the overall posterior spindle displacement that leads to the asymmetric division of the wild-type one-cell stage embryo. In separate work, we analysed a locus called zyg-8, which plays a key role in ensuring proper spindle positioning. Our data show that zyg-8 is required to promote microtubule growth and/or stability during anaphase. We identified the molecular nature of the zyg-8 locus in the course of a large-scale RNAi-based functional genomics screen. ZYG-8 harbours two notable protein domains: a Ca2+/calmodulin-dependent kinase domain, and a domain related to doublecortin, a human microtubule-associated protein involved in neuronal migration.

Large-scale chromatin fibers of living cells display a discontinuous functional organization.

We investigated the chromatin organization of living cells with a combination of recently developed approaches for histone and DNA labeling. Nucleosomal DNA was labeled with a histone H2B-GFP (green fluorescent protein) fusion protein and the chromatin organization of living HeLa cells was analyzed by high resolution confocal microscopy. Within the perinuclear and perinucleolar regions chromatin was organized into large-scale fibers of 2 to 8 microm in length and 300 to 500 nm in diameter. Within the nuclear interior we observed similar large-scale fibers, but in addition focal as well as diffuse forms of organization. Comparison with standard labeling and detection procedures revealed major differences in the chromatin organization observed. Chromatin organization revealed by the distribution of histone H2B-GFP was directly compared with the functional organization of chromatin by Cy3-dUTP labeling of DNA replicating at a specific time. DNA regions replicating at a specific time display characteristic physical and functional properties. Analysis of Cy3-labeled foci revealed that they are associated with all three forms of chromatin organization (fibrillar, focal and diffuse). In particular, Cy3-labeled foci appeared as discontinuous regions of large-scale fibers. These results demonstrate that large-scale chromatin fibers have discontinuous functional characteristics.

Quantitative ER <--> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells.

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/- 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF(4)(-) treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.

Optical trapping of dielectric particles in arbitrary fields.

We present a new method to calculate trapping forces of dielectric particles with diameters D < or = lambda in arbitrary electromagnetic, time-invariant fields. The two components of the optical force, the gradient force and the scattering force, are determined separately. Both the arbitrary incident field and the scatterer are represented by plane-wave spectra. The scattering force is determined by means of the momentum transfer in either single- or double-scattering processes. Therefore the second-order Born series is evaluated and solved in the frequency domain by Ewald constructions. Numerical results of our two-force-component approach and an established calculation method are compared and show satisfying agreement. Our procedure is applied to investigate axial trapping by focused waves experiencing effects of aperture illumination and refractive-index mismatch.

Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo.

Cell divisions that create daughter cells of different sizes are crucial for the generation of cell diversity during animal development. In such asymmetric divisions, the mitotic spindle must be asymmetrically positioned at the end of anaphase. The mechanisms by which cell polarity translates to asymmetric spindle positioning remain unclear. Here we examine the nature of the forces governing asymmetric spindle positioning in the single-cell-stage Caenorhabditis elegans embryo. To reveal the forces that act on each spindle pole, we removed the central spindle in living embryos either physically with an ultraviolet laser microbeam, or genetically by RNA-mediated interference of a kinesin. We show that pulling forces external to the spindle act on the two spindle poles. A stronger net force acts on the posterior pole, thereby explaining the overall posterior displacement seen in wild-type embryos. We also show that the net force acting on each spindle pole is under control of the par genes that are required for cell polarity along the anterior-posterior embryonic axis. Finally, we discuss simple mathematical models that describe the main features of spindle pole behaviour. Our work suggests a mechanism for generating asymmetry in spindle positioning by varying the net pulling force that acts on each spindle pole, thus allowing for the generation of daughter cells with different sizes.

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells.

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

The BioImage Database Project: organizing multidimensional biological images in an object-relational database.

The BioImage Database Project collects and structures multidimensional data sets recorded by various microscopic techniques relevant to modern life sciences. It provides, as precisely as possible, the circumstances in which the sample was prepared and the data were recorded. It grants access to the actual data and maintains links between related data sets. In order to promote the interdisciplinary approach of modern science, it offers a large set of key words, which covers essentially all aspects of microscopy. Nonspecialists can, therefore, access and retrieve significant information recorded and submitted by specialists in other areas. A key issue of the undertaking is to exploit the available technology and to provide a well-defined yet flexible structure for dealing with data. Its pivotal element is, therefore, a modern object relational database that structures the metadata and ameliorates the provision of a complete service. The BioImage database can be accessed through the Internet.

Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light.

A quadrant photodiode placed in the back-focal plane of the microscope of a laser trap provides a high-resolution position sensor. We show that in addition to the lateral displacement of a trapped sphere, its axial position can be measured by the ratio of the intensity of scattered laser light to the total amount of the light reaching the detector. The addition of the axial information offers true three-dimensional position detection in solution, creating, together with a position control, a photonic force microscope with nanometer spatial and microsecond temporal resolution. The measured position signals are explained as interference of the unscattered trapping laser beam with the laser light scattered by the trapped bead. Our model explains experimental data for trapped particles in the Rayleigh regime (radius a <0.2lambda) for displacements up to the focal dimensions. The cross-talk between the signals in the three directions is explained and it is shown that this cross-talk can be neglected for lateral displacements smaller than 75 nm and axial displacements below 150 nm. The advantages of three-dimensional single-particle tracking over conventional video-tracking are shown through the example of the diffusion of the GPI-anchored membrane protein Thy1.1 on a neurite.

Organising multi-dimensional biological image information: the BioImage Database.

Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional image information. At the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques, is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context that work on BioImage started two years ago, and that its first version is now presented here. In essence, BioImage is a database specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of BioImage can be accessed at http://www. bioimage.org and http://www-embl.bioimage.org

Resolution improvement by nonconfocal theta microscopy.

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

Recycling of golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering.

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11-amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme beta1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2-GFP and GalT-GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2-VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1pdn) blocking ER export. Sar1pdn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2-VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1pdn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.

Structure and dynamics of human interphase chromosome territories in vivo.

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5-10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.