Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.     

Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure

The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer.     

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.     

mRNA localization: motile RNA, asymmetric anchors

Techniques to label mRNA with green fluorescent protein (GFP) have provided the first real-time images of RNA motility in live yeast cells. Genetic screens for factors responsible for mRNA asymmetry (e. g. SHE genes) in yeast identified type V myosin among other proteins. Analysis of mRNA movement in various she mutants revealed the role of motor proteins in long-range transport, factors for particle formation, and cortical anchors for docking the mRNA.     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Individual cell motility studied by time-lapse video recording: influence of experimental conditions

BACKGROUND: Eukaryotic cell motility plays a key role during development, wound healing, and tumour invasion. Computer-assisted image analysis now makes it a realistic task to quantify individual cell motility of a large number of cells. However, the influence of culture conditions before and during measurements has not been investigated systematically. METHODS: We have evaluated intraassay and interassay variations in determinations of cellular speed of fibroblastoid L929 cells and investigated the effects of a series of physical and biological parameters on the motile behavior of this cell line. Cellular morphology and organization of filamentous actin were assessed by means of phase-contrast and confocal laser scanning microscopy and compared to the corresponding motility data. RESULTS: Cell dissociation procedure, seeding density, time of cultivation, and substrate concentration were shown to affect cellular speed significantly. pH and temperature of the medium most profoundly influenced cell motility and morphology. Thus, the mean cell speed was 40% lower at pH 7.25 than at pH 7.6; at 29 degrees C, it was approximately four times lower than at 39 degrees C. CONCLUSION: Of the parameters evaluated, cell motility was most strongly affected by changes in pH and temperature. In general, changes in cell speed were accompanied by alterations in cell morphology and organization of filamentous actin, although no consistent phenotypic characteristics could be demonstrated for cells exhibiting high cell speed.     

Gene marking of human neural stem/precursor cells using green fluorescent proteins

BACKGROUND: Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. METHODS: hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. RESULTS: Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. CONCLUSIONS: Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of hNSCs, and is highly useful in vivo. The nature, properties, and possible side effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.     

Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein

E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.     

Cytology of fungal pathogens and plant-host interactions

Imaging plays a unique role in fungal cell biology and phytopathology by allowing for the documentation of molecular structure in individual fixed and living cells. Advances in fluorescence laser techniques, including confocal and multiphoton microscopy, are opening new avenues for cellular exploration. These techniques hold tremendous potential for studies of host-pathogen interactions including the use of genetically encoded markers such as green fluorescent protein, in situ hybridization and fluorescence resonance energy transfer.     

Multiplexing clonality: combining RGB marking and genetic barcoding

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.     

Following cell-fate in E. coli after infection by phage lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.     

Green fluorescent protein (GFP)-dependent separation of bacterial ghosts from intact cells by FACS

BACKGROUND: E. coli and Salmonella ghost preparations, produced by applying the PhiX174 protein E-mediated lysis system, contain nonlysed bacteria at a very low percentage. To use the ghosts as vaccines, additional methods have to be identified to remove any viable cell, to end up in totally inactivated ghost fractions. Materials and Methods To increase the purity of ghost fractions, we established a green fluorescent protein (GFP)-dependent "in vivo staining" method to be combined with the E-mediated lysis system. Several gfp expression vectors were constructed, and the corresponding cellular fluorescence was analyzed. Bacterial fluorescence, exclusively preserved in nonlysed cells, was utilized to separate these cells from ghost preparations via flow cytometric sorting. RESULTS: High-level production of GFP prior to induction of the lysis system did not affect bacterial growth rates and caused no inhibitory effects on the subsequent protein E-mediated lysis of the cells. The population of reproductive or inactivated but nonlysed cells was highly fluorescent at mean intensities 215-fold higher than ghosts, which exhibited fluorescence at background level. Fluorescent cells could effectively be separated from ghost preparations via flow cytometric sorting. Cell sorting subsequent to protein E-mediated lysis reduced the number of viable cells within ghost preparations by a factor of 3 x 10(5). CONCLUSIONS: The presented procedure is compatible with the protein E-mediated lysis system, is highly effective in separation of nonlysed fluorescent cells, and may serve as a prototype for ghost-purification in applications where only a minimum number of viable cells within ghost preparations can be tolerated.     

Four-color, 4-D time-lapse confocal imaging of chick embryos

Our understanding of the molecular mechanisms that direct cell motility, cell division, and cell shaping has benefited from innovations in cell labeling and the ability to resolve intracellular dynamics with multispectral, high-resolution imaging. However, due to difficulties with in vivo cell marking and monitoring, most studies have been restricted to fixed tissue or cells in culture. Here, we report the delivery of multiple (up to four), multicolor fluorescent protein (FP) constructs and four-dimensional (4-D), multispectral time-lapse confocal imaging of cell movements in living chick embryos. Cell cytoskeletal components are fluorescently tagged after microinjection and electroporation of a cocktail of FP constructs into specific regions of chick embryos. We tested 11 different FP constructs in various two-, three-, and four-color combinations using multispectral imaging and linear unmixing to limit the crosstalk between different emission spectra. We monitored intracellular dynamics in individual multicolored migrating cells in vivo and developed a set of advantageous imaging parameters for 4-D time-lapse confocal microscopy. We find that the number of four-color labeled cells in a typical embryo is approximately 10% of the total number of fluorescently labeled cells; this value consistently increases showing that approximately 50% of the total labeled cells have only one-color. We find that multicolored cells are photostable for time-lapses of approximately 2-3 h. Thus, cell labeling with up to four FP color schemes combined with multispectral, 4-D confocal time-lapse imaging offers a powerful tool to simultaneously analyze cellular and molecular dynamics during chick embryogenesis.     

Endothelial cell motility is compatible with junctional integrity

Confluent endothelial cells in culture are generally regarded as a model of resting endothelium in blood vessels (i.e., forming junctions at points of cell-cell contact, losing ability to proliferate in response to growth factors, and remaining stationary). However, incompatibility between junctional integrity and endothelial cell motility remains uncertain. The aim of this study was to determine whether endothelial cells (in colonies generated from differentiating embryonic stem cells in contact with OP9 stromal cell layer) have a resting endothelial phenotype (i.e., lack motility). Time-lapse analyses showed that though endothelial cells were connected to each other through adherens junctions and tight junctions, they were moving continuously within the colonies. Endothelial cell movement was accompanied by formation of lamellipodia, which transiently accumulated green fluorescent protein-tagged beta-actin and p41-Arc (a subunit of the actin-related protein 2/3 complex) at their anterior tips, suggesting that the movement is an active behavior of endothelial cells. Endothelial cell-specific expression of yellow fluorescent protein-tagged vascular endothelial-cadherin and claudin-5 revealed that adherens junctions and tight junctions persisted during endothelial cell migration. Furthermore, intercellular junctions underwent dynamic remodeling at the leading edge of moving endothelial cells. These results suggest that endothelial cells can remain highly motile without losing intercellular junctions.     

Centrosome reorientation in wound-edge cells is cell type specific

The reorientation of the microtubule organizing center during cell migration into a wound in the monolayer was directly observed in living wound-edge cells expressing gamma-tubulin tagged with green fluorescent protein. Our results demonstrate that in CHO cells, the centrosome reorients to a position in front of the nucleus, toward the wound edge, whereas in PtK cells, the centrosome lags behind the nucleus during migration into the wound. In CHO cells, the average rate of centrosome motion was faster than that of the nucleus; the converse was true in PtK cells. In both cell lines, centrosome motion was stochastic, with periods of rapid motion interspersed with periods of slower motion. Centrosome reorientation in CHO cells required dynamic microtubules and cytoplasmic dynein/dynactin activity and could be prevented by altering cell-to-cell or cell-to-substrate adhesion. Microtubule marking experiments using photoactivation of caged tubulin demonstrate that microtubules are transported in the direction of cell motility in both cell lines but that in PtK cells, microtubules move individually, whereas their movement is more coherent in CHO cells. Our data demonstrate that centrosome reorientation is not required for directed migration and that diverse cells use distinct mechanisms for remodeling the microtubule array during directed migration.     

Regulation of actin-dependent cytoplasmic motility by type II phytochrome occurs within seconds in Vallisneria gigantea epidermal cells

The effects of light on actin-dependent cytoplasmic motility in epidermal cells of green leaves of the aquatic angiosperm Vallisneria gigantea were investigated quantitatively using a custom-made dynamic image analyzer. Cytoplasmic motility was measured by monitoring changes in the brightness of individual pixels on digitized images taken sequentially under infrared light. Acceleration and deceleration of cytoplasmic motility were regulated photoreversibly by type II phytochrome(s). This phytochrome-dependent induction of cytoplasmic motility did not occur uniformly in cytoplasm but took place as scattered patches in which no particular organelles, including nucleus, existed. The induction became detectable at 2.5 s after the start of irradiation with pulsed red light. In cells exposed to microbeam irradiation, cytoplasmic motility was induced only in sites in the cytoplasm that were irradiated directly, whereas nonirradiated neighboring areas were unaffected. The effect was short-lived, disappearing within a few minutes, and no signal was transmitted from an irradiated cell to its neighbors. Anti-phytochrome antibody-responsive protein(s) was detectable in the leaf extract by immunoblot and zinc blot analyses and in cryosections of the epidermis by immunocytochemistry. Although the phytochrome-dependent cytoplasmic motility was blocked by exogenously applied latrunculin B or cytochalasins, treatment of the dark-adapted cells with Ca(2+)-chelating reagents induced the cytoplasmic motility. We have proposed a model for the phytochrome regulation of cytoplasmic motility as one of the earliest responses to a light stimulus.     

Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts.

To accurately interpret the data from fluorescent proteins as reporters of gene activation within living cells, it is important to understand the kinetics of the degradation of the reporter proteins. We examined the degradation kinetics over a large number (>1,000) of single, living cells from a clonal population of NIH3T3 fibroblasts that were stably transfected with a destabilized, enhanced green fluorescent protein (eGFP) reporter driven by the tenascin-C promoter. Data collection and quantification of the fluorescence protein within a statistically significant number of individual cells over long times (14 h) by automated microscopy was facilitated by culturing cells on micropatterned arrays that confined their migration and allowed them to be segmented using phase contrast images. To measure GFP degradation rates unambiguously, protein synthesis was inhibited with cycloheximide. Results from automated live cell microscopy and image analysis indicated a wide range of cell-to-cell variability in the GFP fluorescence within individual cells. Degradation for this reporter was analyzed as a first order rate process with a degradation half-life of 2.8 h. We found that GFP degradation rates were independent of the initial intensity of GFP fluorescence within cells. This result indicates that higher GFP abundance in some cells is likely due to higher rates of gene expression, because it is not due to systematically lower rates of protein degradation. The approach described in this study will assist the quantification and understanding of gene activity within live cells using fluorescent protein reporters.     

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion

BACKGROUND: Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. METHODS: The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. RESULTS: We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. CONCLUSIONS: These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.