Cell Tracking using a Distributed Algorithm for 3D Image Segmentation

We have developed and tested an automated method for simultaneous 3D tracking of numerous, flourescently-tagged cells. The procedure uses multiple thresholding to segment individual cells at a starting timepoint, and then iteratively applies a template-matching algorithm to locate a particular cell\u27s position at subsequent time points. To speed up the method, we have developed a distributed implementation in which template matching is carried out in parallel on several different server machines. The distributed implementation showed a monotonic decrease in response time with increasing number of servers (up to 15 tested), demonstrating that the tracking algorithm is well suited to parallelization, and that nearly real-time performance could be expected on a parallel processor. Of four different template matching statistics tested for 3D tracking of amebae from the cellular slime mold Dictyostelium discoideum, we found that the automated procedure performed best when using a correlation statistic for matching. Using this statistic, the method achieved a .985% success rate in correctly identifying a cell from one timepoint to the next. This method is now being used regularly for 3D tracking of normal and mutant cells of D. discoideum, and as such provides a means to quantify the motion of many cells within a three-dimensional tissue mass

SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1–conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis

Organization of chromatin and histone modifications at a transcription site

According to the transcription factory model, localized transcription sites composed of immobilized polymerase molecules transcribe chromatin by reeling it through the transcription site and extruding it to form a surrounding domain of recently transcribed decondensed chromatin. Although transcription sites have been identified in various cells, surrounding domains of recently transcribed decondensed chromatin have not. We report evidence that transcription sites associated with a tandem gene array in mouse cells are indeed surrounded by or adjacent to a domain of decondensed chromatin composed of sequences from the gene array. Formation of this decondensed domain requires transcription and topoisomerase IIα activity. The decondensed domain is enriched for the trimethyl H3K36 mark that is associated with recently transcribed chromatin in yeast and several mammalian systems. Consistent with this, chromatin immunoprecipitation demonstrates a comparable enrichment of this mark in transcribed sequences at the tandem gene array. These results provide new support for the pol II factory model, in which an immobilized polymerase molecule extrudes decondensed, transcribed sequences into its surroundings

Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography

A key drug target for malaria has been the detoxification pathway of the iron containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug developmen

Space-time clustering of childhood central nervous system tumours in Yorkshire, UK

<p>Abstract</p> <p>Background</p> <p>We specifically tested the aetiological hypothesis that a factor influencing geographical or temporal heterogeneity of childhood central nervous system (CNS) tumour incidence was related to exposure to a transient environmental agent.</p> <p>Methods</p> <p>Information was extracted on individuals aged 0-14 years, diagnosed with a CNS tumour between the 1st January 1974 and 31st December 2006 from the Yorkshire Specialist Register of Cancer in Children and Young People. Ordnance Survey eight-digit grid references were allocated to each case with respect to addresses at the time of birth and the time of diagnosis, locating each address to within 0.1 km. The following diagnostic groups were specified <it>a priori </it>for analysis: ependymoma; astrocytoma; primitive neuroectodermal tumours (PNETs); other gliomas; total CNS tumours. We applied the <it>K</it>-function method for testing global space-time clustering using fixed geographical distance thresholds. Tests were repeated using variable nearest neighbour (NN) thresholds.</p> <p>Results</p> <p>There was statistically significant global space-time clustering for PNETs only, based on time and place of diagnosis (<it>P </it>= 0.03 and 0.01 using the fixed geographical distance and the variable NN threshold versions of the <it>K</it>-function method respectively).</p> <p>Conclusions</p> <p>There was some evidence for a transient environmental component to the aetiology of PNETs. However, a possible role for chance cannot be excluded.</p

CKAP2 ensures chromosomal stability by maintaining the integrity of microtubule nucleation sites

Integrity of the microtubule spindle apparatus and intact cell division checkpoints are essential to ensure the fidelity of distributing chromosomes into daughter cells. Cytoskeleton-associated protein 2, CKAP2, is a microtubule-associated protein that localizes to spindle poles and aids in microtubule stabilization, but the exact function and mechanism of action are poorly understood. In the present study, we utilized RNA interference to determine the extent to which the expression of CKAP2 plays a role in chromosome segregation. CKAP2-depleted cells showed a significant increase of multipolar mitoses and other spindle pole defects. Notably, when interrogated for microtubule nucleation capacity, CKAP2-depleted cells showed a very unusual phenotype as early as two minutes after release from mitotic block, consisting of dispersal of newly polymerized microtubule filaments through the entire chromatin region, creating a cage-like structure. Nevertheless, spindle poles were formed after one hour of mitotic release suggesting that centrosome-mediated nucleation remained dominant. Finally, we showed that suppression of CKAP2 resulted in a higher incidence of merotelic attachments, anaphase lagging, and polyploidy. Based on these results, we conclude that CKAP2 is involved in the maintenance of microtubule nucleation sites, focusing microtubule minus ends to the spindle poles in early mitosis, and is implicated in maintaining genome stability