The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers.
Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM) that is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovered 30 human and yeast histidine repeat proteins that are specifically modified by polyP. This polyP modification is histidine-dependent and non-covalent in nature, though remarkably, it withstands harsh denaturing conditions - a hallmark of covalent PTMs. We have termed this interaction ionic histidine polyphosphorylation (iH-PPn) to describe its unique PTM-like properties. Importantly, we show that iH-PPn disrupts phase separation and phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting iH-PPn as a potential hitherto unrecognized regulatory mechanism.
For decades the concepts of color breeding have baffled the newcomers to the hobby of breeding canaries. Color breeding is one of the most intriguing and challenging facets of canary breeding. It is probably no harder to breed show quality color-bred canaries than it is to breed show quality type birds.First, it demands a knowledge of canary breeding and maintenance.Second, it demands an understanding of some of the simpler genetic inheritance theories. Neither should scare anyone away from breeding color-bred canaries. The care and breeding habits for the color-bred are so similar to those of the other canaries that information can be found elsewhere under the topic general care.When breeding for color, one whould remember that color-bred canaries have the same feather structure as the type canaries - hard and soft-feathers. Therefore, the practice of breeding a hardfeathered bird to a soft-feathered bird is basic. The color-bred canaries are present in both the lipochrome (clear) or melanin (dark) varieties.The lipochrome birds consist of those birds that are completely void of melanin pigments. These birds display only the basic ground colors of yellow, white, or red-orange. The yellow ground is similar to the ground color of the original wild canary. The white ground is the result of a spontaneous mutation. And the red-orange ground is through hybridization. It is a man-made or induced color.The white grounds are of two distinct types, referred to as dominant white or recessive white. The dominant white gene is dominant to both yellow and redorange. It is a homozygous dominant lethal factor, or to say it simply, if a dominant white is paired to another dominant white, 25% of the young will probably die. A dominant white should be paired to either a yellow or red-orange ground bird. The dominant white shows signs of the other ground color gene by the presence of small amounts of yellow or orange in the primary or outer flight feathers and in the tail feathers. Since the recessive white is not very abundant in most areas, the novice need not fear purchasing one or more by accident. Genetically, the recessive white gene must be present in both parents (either self white or carriers) for a white offspring to appear. These birds are pure white and are genetically recessive to both the yellow and the red-orange ground color. Recessive whites are delicate birds since they have difficulties producing Vitamin A, therefore, they should be reserved for the more knowledgeable color breeders.The red-orange ground canary is the most unique quirk of nature. These birds are the result of the hybridization of the canary (Serinus Canarius) and the Blackheaded Red siskin (Spinus Cucutlatus), which is an inhabitant of Venezuela. It was a one-in-a-million chance that such a hybridization would produce fertile young, but now it is a common occurence. Through many long years of hard work. our predecessors have given us the crimson-like beauties of today.There is only one mutation that affects these three ground colors. It is called ivory. The effect is that it masks or reduces the ground color to approximately one half its normal intensity. It reduces the red-orange almost to pink and the yellow to a very pale and delicate yellow, but it has little or no visual effect on the white ground colors.The main message here is directed to the novice breeder of color-bred canaries. So, let these glimpses of the color breeding fancy stimulate in you a desire to participate and to learn. You should not be afraid of those areas of canary breeding in which you have not been involved. Nor should you let the need of acquiring a knowledge of genetics prevent you from enjoying one of the most intriguing aspects of the fancy. Purchase a pair or two from a competent breeder of color-bred canaries and begin a breeding program of your own. Your knowledge of their genetics will increase much faster as you work and see the results of your pairings. Try it! Color breeding is fun! ,
Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.
The vast majority of cyanophages isolated to date are cyanomyoviruses, a group related to bacteriophage T4. Comparative genome analysis of five cyanomyoviruses, including a newly sequenced cyanophage S-RSM4, revealed a 'core genome' of 64 genes, the majority of which are also found in other T4-like phages. Subsequent comparative genomic hybridization analysis using a pilot microarray showed that a number of 'host' genes are widespread in cyanomyovirus isolates. Furthermore, a hyperplastic region was identified between genes g15-g18, within a highly conserved structural gene module, which contained a variable number of inserted genes that lacked conservation in gene order. Several of these inserted genes were host-like and included ptoX, gnd, zwf and petE encoding plastoquinol terminal oxidase, 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase and plastocyanin respectively. Phylogenetic analyses suggest that these genes were acquired independently of each other, even though they have become localized within the same genomic region. This hyperplastic region contains no detectable sequence features that might be mechanistically involved with the acquisition of host-like genes, but does appear to be a site specifically associated with the acquisition process and may represent a novel facet of the evolution of marine cyanomyoviruses.
Automated high-throughput analysis of single-cell timecourse data presents a major bottleneck in live cell imaging. We present LineageTracker, an ImageJ framework to track expression of fluorescent gene reporters over multiple cell divisions. It is able to perform automatic segmentation and tracking, and allows viewing and editing of tracks. The main feature of the tracking algorithm is a statistical scoring method which takes into account characteristic intensity and size changes to classify dividing and non-dividing cells. By including such dynamic features, the method can identify dividing cells in time series with 30 min frame intervals, and handle large cell displacements between frames. We created a manually validated data set of mouse C2C12 cells expressing a fluorescent protein targeted to the cell nucleus which we will make available for benchmarking different segmentation and tracking methods.
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