Kinetics of microcolony formation of a soil oligotrophic bacterium, Agromonas sp.

Abstract The kinetics of budding/dividing of parent cells at different culture ages, spread on a fresh medium, was formulated by the following model N _t= N _∞ [1 − exp (− λ ( t − t _r)] where N _t is the number of budding/dividing cells in the parent population at time t , N _∞ is the expected number of budding/dividing cells at infinite time, λ is the rate of budding/dividing of parent cells, and t _r is the retardation time. The rate of budding/dividing λ decreased with the increase in the culture age of the parent cell population.     

Mouse embryos exposed to oxygen concentrations that mimic changes in the oviduct and uterus show improvement in blastocyst rate,blastocyst size,and accelerated cell division

After in vivo fertilisation, the preimplantation embryo goes through cleavage during migration along the oviduct in mammals or the fallopian tube in a woman and ends up inside the uterus. This study investigates the effect of a protocol aimed at closely reproducing that natural oxygen concentration in the oviduct (7 % O₂ from day 1 to day 3 and 2 % from day 3 to day 5), in contrast to the concentrations (5 % or 20 %) widely used in practice in ART using morphokinetic. Female mice (BI6/CBAca) were sacrificed, and zygotes were isolated 20 h after mating and randomly allocated to three parallel groups, which were grown under high atmospheric, low, or sequential oxygen concentrations. Zygotes were cultured in GTL medium (Vitrolife) and observed by the Primovision time-lapse system. Blastocyst rate at 120 h in the sequential group (91.3 %) was significantly increased over the high (76.3 %) and low (74.4 %) groups. Blastocyst size was also enlarged in the sequential group compared to the high and low groups. Moreover, cell division in the sequential group was significantly faster at almost every cleavage stage than it was in the other groups. Notably, the duration of the interims between stages also differed significantly between the groups. This study demonstrated that, in comparison to routinely used high or low oxygen conditions, oxygen concentrations mimicking changes in the oviduct and uterus significantly improve the blastocyst rate and size and accelerate cell division at several stages as well as the interims between cleavage events.     

Analysis of tissue flow patterns during primitive streak formation in the chick embryo

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.     

Using time-lapse digital video recording for a nesting study of birds of prey

Remote photography using various photo, movie or video devices has been employed in numerous studies in wildlife research during the last 50 years. Given the rapid advances in digital technologies, digital video and photo techniques are becoming more common in use, and publications that introduce a new method or equipment for video surveillance in wildlife research (and in ornithological studies particularly) are appearing almost every year. However, still no special guide to the great variety of equipment and methods is available, and the choice and use of suitable gear for scientific purposes may be difficult for non-specialists. In this paper, we review the most common surveillance techniques used in today’s nest studies, as well as the most essential properties of image recording equipment. We also describe the digital video recording technique, which we used for observations of raptor nests, and summarise our experience of its operation. As an example of the obtained data, we present the timing of prey deliveries of goshawks and common buzzards.     

Automated tracking of unmarked cells migrating in three-dimensional matrices applied to anti-cancer drug screening

In oncology, combating the spread of tumor cells is a clinical need which currently remains unsatisfied. Identifying anti-migratory compounds usually requires in vitro screening of a large number of molecules. Efficient and realistic (i.e., preferably 3D) in vitro tests are thus required in order to quantify the anti-migratory effects of anti-cancer drugs. To remain compatible with high-throughput screening, we focus on assays where unlabeled cells are migrating in 3D transparent gels and are observed under time-lapse 3D phase-contrast microscopy. In this context, we present a method for automatically tracking cells that combines a template matching preprocessing step with a mean-shift process. The preprocessing step consists in performing a correlation of a cell template with each observed volume in order to provide a phase-contrast artifact-free volume where the cells appear as correlation peaks surrounded by smooth gradients. This transformation enables the cells to be efficiently tracked by a mean-shift process. Robustness and efficiency of this approach are qualitatively and quantitatively shown in various experiments. Finally, we successfully applied our method to the quantitative characterization of the anti-migratory impact of cytochalasin-D on cancer cells. In conclusion, our method can efficiently be used for drug screening aiming to evidence drug-induced effects on cell migration in 3D transparent environments, such as matrix gels.     

Vascular sprout formation entails tissue deformations and VE-cadherin-dependent cell-autonomous motility

Embryonic and fetal vascular sprouts form within constantly expanding tissues. Nevertheless, most biological assays of vascular spouting are conducted in a static mechanical milieu. Here we study embryonic mouse allantoides, which normally give raise to an umbilical artery and vein. However, when placed in culture, allantoides assemble a primary vascular network. Unlike other in vitro assays, allantoic primordial vascular cells are situated on the upper surface of a cellular layer that is engaged in robust spreading motion. Time-lapse imaging allows quantification of primordial vascular cell motility as well as the underlying mesothelial tissue motion. Specifically, we calculate endothelial cell-autonomous motion by subtracting the tissue-level mesothelial motion from the total endothelial cell displacements. Formation of new vascular polygons is hindered by administration of function-blocking VE-cadherin antibodies. Time-lapse recordings reveal that (1) cells at the base of sprouts normally move distally “over” existing sprout cells to form new tip-cells; and (2) loss of VE-cadherin activity prevents this motile behavior. Thus, endothelial cell-cell-adhesion-based motility is required for the advancement of vascular sprouts within a moving tissue environment. To the best of our knowledge, this is the first study that couples endogenous tissue dynamics to assembly of vascular networks in a mammalian system.     

Videomicroscopic extraction of specific information on cell proliferation and migration in vitro

In vitro cell imaging is a useful exploratory tool for cell behavior monitoring with a wide range of applications in cell biology and pharmacology. Combined with appropriate image analysis techniques, this approach has been shown to provide useful information on the detection and dynamic analysis of cell events. In this context, numerous efforts have been focused on cell migration analysis. In contrast, the cell division process has been the subject of fewer investigations. The present work focuses on this latter aspect and shows that, in complement to cell migration data, interesting information related to cell division can be extracted from phase-contrast time-lapse image series, in particular cell division duration, which is not provided by standard cell assays using endpoint analyses. We illustrate our approach by analyzing the effects induced by two sigma-1 receptor ligands (haloperidol and 4-IBP) on the behavior of two glioma cell lines using two in vitro cell models, i.e., the low-density individual cell model and the high-density scratch wound model. This illustration also shows that the data provided by our approach are suggestive as to the mechanism of action of compounds, and are thus capable of informing the appropriate selection of further time-consuming and more expensive biological evaluations required to elucidate a mechanism.     

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recording mitotic chromosome cycle induction in living plant cells

Summary The presence of a prophase nucleus inHaemanthus endosperm happens to trigger the break down of the nuclear envelope in any interphase nucleus, located in its close proximity. Besides, chromosomes in the interphase nucleus start condensing gradually for the initial breaking point which is the nearest point to the prophase. The observation suggest the diffusion of an inducer, whose progression has been recorded to occur at a rate of 1.1 m/min.