When the Genome Plays Dice: Circumvention of the Spindle Assembly Checkpoint and Near-Random Chromosome Segregation in Multipolar Cancer Cell Mitoses

Background: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes. Cancer cells, however, occasionally divide into three or more directions. Such multipolar mitoses have been proposed to generate genetic diversity and thereby contribute to clonal evolution. However, this notion has been little validated experimentally.Principal Findings: Chromosome segregation and DNA content in daughter cells from multipolar mitoses were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells. The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells. Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition. The multipolar metaphase-anaphase transition was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the normal separase activity cycle. Centromeric AURKB and MAD2 foci were observed frequently to remain on the centromeres of multipolar ana-telophase chromosomes, indicating that multipolar mitoses were able to circumvent the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase. Accordingly, scoring the distribution of individual chromosomes in multipolar daughter nuclei revealed a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister chromatids to the daughter cells.Conclusion: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells. Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations

Multipolar Spindle Pole Coalescence Is a Major Source of Kinetochore Mis-Attachment and Chromosome Mis-Segregation in Cancer Cells

Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed γ-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells

Simplified vehicle calibration using multilinear constraints

An Autonomously Guided Vehicle using both odometry and visual data for navigation needs calibration parameters. These include camera placement as well as parameters relating odometry to vehicle motion. Calibration of these parameters is related to the Hand-Eye calibration problem. Instead of using a calibration target or trying to solve for structure and motion a novel method using the continuous multilinear constraint to test parameter combinations is proposed. A low order polynomial target function is calculated in linear time over the sample size resulting in very fast iterations in the optimisation step. The method is tested on simulated data and increased sample size improves the parameter estimates

A Cost-Effective Automatic 3D Reconstruction Pipeline for Plants Using Multi-view Images

Plant phenotyping involves the measurement, ideally objectively, of characteristics or traits. Traditionally, this is either limited to tedious and sparse manual measurements, often acquired destructively, or coarse image-based 2D measurements. 3D sensing technologies (3D laser scanning, structured light and digital photography) are increasingly incorporated into mass produced consumer goods and have the potential to automate the process, providing a cost-effective alternative to current commercial phenotyping platforms. We evaluate the performance, cost and practicability for plant phenotyping and present a 3D reconstruction method from multi-view images acquired with a domestic quality camera. This method consists of the following steps: (i) image acquisition using a digital camera and turntable; (ii) extraction of local invariant features and matching from overlapping image pairs; (iii) estimation of camera parameters and pose based on Structure from Motion(SFM); and (iv) employment of a patch based multi-view stereo technique to implement a dense 3D point cloud. We conclude that the proposed 3D reconstruction is a promising generalized technique for the non-destructive phenotyping of various plants during their whole growth cycles

Bundle Adjustment for Stereoscopic 3D

Abstract. The recent resurgence of stereoscopic 3D films has triggered a high demand for post-processing tools for stereoscopic image sequences. Camera motion estimation, also known as structure-from-motion (SfM) or match-moving, is an essential step in the post-processing pipeline. In order to ensure a high accuracy of the estimated camera parameters, a bundle adjustment algorithm should be employed. We present a new stereo camera model for bundle adjustment. It is designed to be applicable to a wide range of cameras employed in today’s movie productions. In addition, we describe how the model can be integrated efficiently into the sparse bundle adjustment framework, enabling the processing of stereoscopic image sequences with traditional efficiency and improved accuracy. Our camera model is validated by synthetic experiments, on rendered sequences, and on a variety of real-world video sequences.

Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase–anaphase transition and thereby physiologically satisfy the SAC