Comparison of Interactions Between Control and Mutant Macrophages

This paper presents a preliminary study on macrophages migration in Drosophila embryos, comparing two types of cells. The study is carried out by a framework called macrosight which analyses the movement and interaction of migrating macrophages. The framework incorporates a segmentation and tracking algorithm into analysing motion characteristics of cells after contact. In this particular study, the interactions between cells is characterised in the case of control embryos and Shot3 mutants, where the cells have been altered to suppress a specific protein, looking to understand what drives the movement. Statistical significance between control and mutant cells was found when comparing the direction of motion after contact in specific conditions. Such discoveries provide insights for future developments in combining biological experiments to computational analysis

Shape analysis and tracking of migrating macrophages

Cell migration is important in many human processes of development and disease. In Cancer, migration can be related to metastasis or cell defects. A precise analysis of the cell shapes in biological studies could lead to insights about migration. Therefore, this paper describes an algorithm to iteratively segment, track and analyse the shape of macrophages from fluorescent microscopy image sequences. This process allows observation of shape variations as the cells migrate. The algorithm identifies and separates overlapping and non-overlapping cells, then for the non-overlapping cases analyses the shape and extracts a series of measurements, including the number of "corner" or pointy edges through a multiscale angle variation matrix, anglegram. The shape evolution algorithm was tested on fluorescently labelled macrophages observed on embryos of Drosophila melanogaster

Analysis of the Interactions of Migrating Macrophages

Understanding the migrating patterns of cells in the immune system is of great importance; especially the changes of direction and its cause. For macrophages and other immune cells, excessive migration could be related to autoimmune diseases and cancer. In this work, an algorithm to analyse the change in direction of cells before and after they interact with another cell is proposed. The main objective is to provide insights into the notion that interactions between cell structures appear to anticipate migration. Such interactions are determined when the cells overlap and form clumps of two or more cells. The algorithm integrates a segmentation technique capable of detecting overlapping cells and a tracking framework into a tool for the analysis of the trajectories of cells before and after they overlap. The preliminary results show promise into the analysis and the hypothesis proposed, and it lays the ground work for further developments

Segmentation of Overlapping Macrophages Using Anglegram Analysis

This paper describes the automatic segmentation of overlapping cells through different algorithms. As the first step, the algorithm detects junctions between the boundaries of overlapping objects based on the angles between points of the overlapping boundary. For this purpose, a novel 2D matrix with multiscale angle variation is introduced, i.e anglegram. The anglegram is used to find junctions of overlapping cells. The algorithm to retrieve junctions from the boundary was tested and validated with synthetic data and fluorescently labelled macrophages observed on embryos of Drosophila melanogaster. Then, four different segmentation techniques were evaluated: (i) a Voronoi partition based on the nuclei positions, (ii) a slicing method, which joined the clumps together (junction slicing), (iii) a partition based on the following of the edges from the junctions (edge following), and (iv) a custom self-organising map to fit to the area of overlap between the cells. Only (ii)-(iv) were based on the junctions. The segmentation results were compared based on precision, recall and Jaccard similarity. The algorithm that reported the best segmentation was the junction slicing

Comparative Study of Contact Repulsion in Control and Mutant Macrophages Using a Novel Interaction Detection

In this paper, a novel method for interaction detection is presented to compare the contact dynamics of macrophages in the Drosophila embryo. The study is carried out by a framework called macrosight, which analyses the movement and interaction of migrating macrophages. The framework incorporates a segmentation and tracking algorithm into analysing the motion characteristics of cells after contact. In this particular study, the interactions between cells is characterised in the case of control embryos and Shot mutants, a candidate protein that is hypothesised to regulate contact dynamics between migrating cells. Statistical significance between control and mutant cells was found when comparing the direction of motion after contact in specific conditions. Such discoveries provide insights for future developments in combining biological experiments with computational analysis

GliaMorph: A modular image analysis toolkit to quantify Müller glial cell morphology

Cell morphology is critical for all cell functions. This is particularly true for glial cells as they rely on complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this, we developed the image analysis pipeline "GliaMorph". GliaMorph is a modular analysis toolkit developed to perform (i) image pre-processing, (ii) semi-automatic region-of-interest (ROI) selection, (iii) apicobasal texture analysis, (iv) glia segmentation, and (v) cell feature quantification. Müller Glia (MG) have a stereotypic shape linked to their maturation and physiological status. We here characterized MG on three levels, including (a) global image-level, (b) apicobasal texture, and (c) regional apicobasal vertical-to-horizontal alignment. Using GliaMorph we quantified MG development on a global and single-cell level, showing increased feature elaboration and subcellular morphological rearrangement in the zebrafish retina. As proof-of-principle, we analysed expression changes in a mouse glaucoma model, identifying subcellular protein localization changes in MG. Together, GliaMorph enables an in-depth understanding of MG morphology in the developing and diseased retina

CCL2 and CCR2 regulate pain-related behaviour and early gene expression in post-traumatic murine osteoarthritis but contribute little to chondropathy

SummaryObjectiveThe role of inflammation in structural and symptomatic osteoarthritis (OA) remains unclear. One key mediator of inflammation is the chemokine CCL2, primarily responsible for attracting monocytes to sites of injury. We investigated the role of CCL2 and its receptor CCR2 in experimental OA.DesignOA was induced in 10 weeks old male wild type (WT), Ccl2−/− and Ccr2−/− mice, by destabilisation of the medial meniscus (DMM). RNA was extracted from whole joints at 6 h and 7 days post-surgery and examined by reverse transcription polymerase chain reaction (RT-PCR). Gene expression changes between naïve and DMM-operated mice were compared. Chondropathy scores, from mice at 8, 12, 16 and 20 weeks post DMM were calculated using modified Osteoarthritis Research Society International (OARSI) grading systems. Changes in hind paw weight distribution, as a measure of pain, were assessed by Linton incapacitance.ResultsAbsence of CCL2 strongly suppressed (>90%) selective inflammatory response genes in the joint 6 h post DMM, including arginase 1, prostaglandin synthase 2, nitric oxide synthase 2 and inhibin A. IL6, MMP3 and tissue inhibitor of metalloproteinase 1 were also significantly suppressed. Similar trends were also observed in the absence of CCR2. A lower average chondropathy score was observed in both Ccl2−/− and Ccr2−/− mice at 12, 16 and 20 weeks post DMM compared with WT mice, but this was only statistically significant at 20 weeks in Ccr2−/− mice. Pain-related behaviour in Ccl2−/− and Ccr2−/− mice post DMM was delayed in onset.ConclusionThe CCL2/CCR2 axis plays an important role in the development of pain in murine OA, but contributes little to cartilage damage

Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration

Aa robust inflammatory response to tissue damage and infection is conserved across almost all animal phyla. Neutrophils and macrophages, or their equivalents, are drawn to the wound site where they engulf cell and matrix debris and release signals that direct components of the repair process. This orchestrated cell migration is clinically important, and yet, to date, leukocyte chemotaxis has largely been studied in vitro. Here, we describe a genetically tractable in vivo wound model of inflammation in the Drosophila melanogaster embryo that is amenable to cinemicroscopy. For the first time, we are able to examine the roles of Rho-family small GTPases during inflammation in vivo and show that Rac-mediated lamellae are essential for hemocyte motility and Rho signaling is necessary for cells to retract from sites of matrix– and cell–cell contacts. Cdc42 is necessary for maintaining cellular polarity and yet, despite in vitro evidence, is dispensable for sensing and crawling toward wound cues

Protocol for intervention-free quantification of protein turnover rate by steady-state modeling

Protein turnover rate is difficult to obtain experimentally. This protocol shows how to mathematically model turnover rates in an intervention-free manner given the ability to quantify mRNA and protein expression from initiation to homeostasis. This approach can be used to calculate production and degradation rates and to infer protein half-life. This model was successfully employed to quantify turnover during Drosophila embryogenesis, and we hypothesize that it will be applicable to diverse in vivo or in vitro systems

Mechanisms and in vivo functions of contact inhibition of locomotion

Contact inhibition of locomotion (CIL) is a process whereby a cell ceases motility or changes its trajectory upon collision with another cell. CIL was initially characterized more than half a century ago and became a widely studied model system to understand how cells migrate and dynamically interact. Although CIL fell from interest for several decades, the scientific community has recently rediscovered this process. We are now beginning to understand the precise steps of this complex behaviour and to elucidate its regulatory components, including receptors, polarity proteins and cytoskeletal elements. Furthermore, this process is no longer just in vitro phenomenology; we now know from several different in vivo models that CIL is essential for embryogenesis and in governing behaviours such as cell dispersion, boundary formation and collective cell migration. In addition, changes in CIL responses have been associated with other physiological processes, such as cancer cell dissemination during metastasis