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1 -ABSTRACTDigital light microscopy provides powerful tools for quantitatively probing the real-time dynamics of subcellular structures. While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). Keeping notes on microscopy experiments and quality control procedures ought to be straightforward, as the microscope is a machine whose components are defined and the performance measurable. Nevertheless, to this date, no universally adopted community-driven specifications exist that delineate the required information about the microscope hardware and acquisition settings (i.e., microscopy “data provenance” metadata) and the minimally accepted calibration metrics (i.e., microscopy quality control metadata) that should be automatically recorded by both commercial microscope manufacturers and customized microscope developers. In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To add to the confusion, microscopy experiments vary greatly in aim and complexity, ranging from purely descriptive work to complex, quantitative and even sub-resolution studies that require more detailed reporting and quality control measures.To solve this problem, the4D Nucleome Initiative (4DN) (1, 2) Imaging Standards Working Group (IWG), working in conjunction with theBioImagingNorthAmerica (BINA) Quality Control and Data Management Working Group (QC-DM-WG) (3), here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a revision of the Core of the Open Microscopy Environment (OME) Data Model, which forms the basis for the widely adopted Bio-Formats library (4–6), accompanied by a suite of three extensions, each with three tiers, allowing the classification of imaging experiments into levels of increasing imaging and analytical complexity (7, 8). Hence these specifications not only provide an OME-based comprehensive set of metadata elements that should be recorded, but they also specify which subset of the full list should be recorded for a given experimental tier. In order to evaluate the extent of community interest, an extensive outreach effort was conducted to present the proposed metadata specifications to members of several core-facilities and international bioimaging initiatives including theEuropeanLightMicroscopyInitiative (ELMI),GlobalBioImaging (GBI), andEuropeanMolecularBiologyLaboratory (EMBL) -EuropeanBioinformaticsInstitute (EBI). Consequently, close ties were established between our endeavour and the undertakings of the recently establishedQUAlity Assessment andREProducibility for Instruments and Images inLightMicroscopy global community initiative (9). As a result this flexible 4DN-BINA-OME (NBO namespace) framework (7, 8) represents a turning point towards achieving community-driven Microscopy Metadata standards that will increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of different datasets, experimental setups, and assays, and it demonstrates the method for future extensions. Such universally accepted microscopy standards would serve a similar purpose as the Encode guidelines successfully adopted by the genomic community (10, 11). The intention of this proposal is therefore to encourage participation, critiques and contributions from the entire imaging community and all stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders.

The community-driven initiative Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy (QUAREP-LiMi) wants to improve reproducibility for light microscopy image data through quality control (QC) management of instruments and images. It aims for a common set of QC guidelines for hardware calibration and image acquisition, management and analysis.

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.

The Woronin body (WB) is a peroxisome-related organelle that is centered on a crystalline core of the HEX-1 protein, which functions to seal septal pores of filamentous ascomycetes in response to cellular damage. Here, we investigate the cellular and genetic control of WB-formation and show that polarized hex-1 gene expression determines WB-biogenesis at the growing hyphal apex. We find that intron splicing is coupled to efficient hex-1 gene expression and strikingly, when the yellow fluorescent protein was expressed from hex-1 regulatory sequences, we observed a fluorescent gradient that was maximal in apical cells. Moreover, endogenous hex-1 transcripts were specifically enriched at the leading edge of the fungal colony, whereas other transcripts accumulated in basal regions. Time-lapse confocal microscopy showed that HEX-1 crystals normally formed in the vicinity of the hyphal apex in large peroxisomes, which matured and were immobilized at the cell periphery as cells underwent septation. When the hex-1 structural gene was expressed from regulatory sequences of an abundant, basally localized transcript, WB-core formation was redetermined to basal regions of the colony, and these strains displayed loss-of-function phenotypes specifically in apical hyphal compartments. These results show that apically localized gene expression is a key determinant of spatially restricted WB-assembly. We suggest that this type of regulation may be widely used to determine cellular activity in apical regions of the fungal hypha.

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of two families, desmocollins and desmogleins. Three isoforms of each are expressed in a tissue-specific and developmentally regulated pattern. In bovine nasal epidermis, the three desmocollin (Dsc) isoforms are expressed in overlapping domains; Dsc3 expression is strongest in the basal layer, while Dsc2 and Dsc1 are strongly expressed in the suprabasal layers. Herein we have investigated whether different isoforms are assembled into the same or distinct desmosomes by performing double immunogold labeling using isoform-specific antibodies directed against Dsc1 and Dsc3. The results show that individual desmosomes harbor both isoforms in regions where their expression territories overlap. Quantification showed that the ratio of the proteins in each desmosome altered gradually from basal to immediately suprabasal and upper suprabasal layers, labeling for Dsc1 increasing and Dsc3 decreasing. Thus desmosomes are constantly modified as cells move up the epidermis, with continuing turnover of the desmosomal glycoproteins. Statistical analysis of the quantitative data showed a possible relationship between the distributions of the two isoforms. This gradual change in desmosomal composition may constitute a vertical adhesive gradient within the epidermis, having important consequences for cell positioning and differentiation.

The epidermal blistering disease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130 × 10(3) M(r) polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.

PNC-27 anti-cancer peptide, derived from the MDM2 binding site of p53 and linked to a membrane residency peptide (MRP), has been shown to cause necrosis of cancer cells without affecting untransformed cells. PNC-27 was also able to eradicate pancreatic tumor xenografts in mice (Michl et al, Int. J of Cancer, 2006). Recently, we have identified the mechanism of action of this peptide as due to formation of oligomeric pores in the plasma membrane (PM) of cancer cells but not in the PM of untransformed cells. The mechanism of pore-formation by PNC-27 closely resembles the pore-formation process by streptolysin-O, melittin and similar pore-forming agents. We have shown MDM2 as a targeting molecule that leads to PNC-27 selectivity towards cancer cells by its mis-localization to cancer cell PM (Sarafraz-Yazdi et al, PNAS, in press). Examining purified PM of a variety of cancer cells by immunoblotting, we now provide evidence for multiple MDM2 protein variants, and that were absent in the PM of normal untransformed cells. To confirm the purity of the isolated PM, fractions were also immunoblotted for the PM markers Na+/K+-ATPase, E-Cadherin and ß-Catenin, all of which were enriched in the PM fractions. In contrast, Abs against membrane markers specific for intracellular organelles, COX IV and Cytochrome C for mitochondrial membrane, showed no reaction in the PM fractions while they reacted with total membrane fractions. Three protein variants of MDM2 with MW of 27kD, 40kD and 57kD were consistently expressed in the PM of human and rat pancreatic cancer cells, human melanoma, breast cancer. We also confirmed these observations in isolated PM from freshly obtained primary ovarian tumors from human patients with aggressive tumor. Remarkably, no MDM2 variant was detectable in the PM fractions of primary human fibroblasts and untransformed pancreatic HPNE cells. Our finding of differently sized MDM2 variants complies with other, previously reported MDM2 protein variants. Of the different MDM2 mRNA splice variants published, 5 have been shown to be translated into protein including proteins of 27kD, 40kD and 57kD. Our present study demonstrates the localization of the 3 variant proteins in the PM of cancer cells, suggesting a possible role for these membrane-localized MDM2 variant in the action of PNC-27. The notion of this specific MDM2-PNC-27 interaction at the PM level was strongly supported by the effective competition by a monoclonal MDM2-specific Ab, reducing PNC-27-mediated cytotoxicity by >80% as measured by LDH cytotoxicity assay and propidium iodide staining of the nuclei of dead cells in real-time spinning disc confocal microscopy. These findings for the first time demonstrate not only the novel localization of different MDM2 variants in PM of different cancer cells but they also suggest a potentially wider applicability of PNC-27 as a novel selective anti-cancer drug for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5770.