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

Shape Analysis and Tracking of Migrating Macrophages

This work describes an algorithm to observe cell shape variation associated with migration. The algorithm iteratively segments, tracks and analyses the shape of macrophages in Drosophila melanogaster embryos. Analysis of shape, including the number of corners or pointy edges, rely on a novel approach to finding junctions, the anglegram matrix. The anglegram [1] IS a multiscale angle variation 2D matrix. It Iis constructed by calculating inner point angles alongside the boundaries of an object

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

Cortical sinus probing, S1P1-dependent entry and flow-based capture of egressing T cells.

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in sphingosine 1-phosphate receptor type 1 probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on sphingosine 1-phosphate receptor type 1, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph

The multiple faces of leukocyte interstitial migration

Spatiotemporal control of leukocyte dynamics within tissues is critical for successful innate and adaptive immune responses. Homeostatic trafficking and coordinated infiltration into and within sites of inflammation and infection rely on signaling in response to extracellular cues that in turn controls a variety of intracellular protein networks regulating leukocyte motility, migration, chemotaxis, positioning, and cell–cell interaction. In contrast to mesenchymal cells, leukocytes migrate in an amoeboid fashion by rapid cycles of actin polymerization and actomyosin contraction, and their migration in tissues is generally referred to as low adhesive and nonproteolytic. The interplay of actin network expansion, contraction, and adhesion shapes the exact mode of amoeboid migration, and in this review, we explore how leukocyte subsets potentially harness the same basic biomechanical mechanisms in a cell-type-specific manner. Most of our detailed understanding of these processes derives from in vitro migration studies in three-dimensional gels and confined spaces that mimic geometrical aspects of physiological tissues. We summarize these in vitro results and then critically compare them to data from intravital imaging of leukocyte interstitial migration in mouse tissues. We outline the technical challenges of obtaining conclusive mechanistic results from intravital studies, discuss leukocyte migration strategies in vivo, and present examples of mode switching during physiological interstitial migration. These findings are also placed in the context of leukocyte migration defects in primary immunodeficiencies. This overview of both in vitro and in vivo studies highlights recent progress in understanding the molecular and biophysical mechanisms that shape robust leukocyte migration responses in physiologically complex and heterogeneous environments

Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay

The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment

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

A dual role for the βPS integrin myospheroid in mediating Drosophila embryonic macrophage migration

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main βPS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards wounds.This work was supported by the Spanish Ministerio de Ciencia y Tecnología [grant numbers BFU2010-16669 and CSD-2007-00008 to M.D.M.-B.] and by a Wellcome Trust Senior Research Fellowship [grant number 090899/Z/09/Z to W.W.]. R.R. was supported by the DFG as project B11 of the SFB 446 ‘Zellverhalten der Eukaryoten’]; the EMBO Young Investigator Programme [a Formación de. Personal Investigador studentship from the Spanish Ministerio de Ciencia y Tecnología [to B.J.S.-S.]. and an MRC Doctoral Training Grant to K.C.Peer reviewe

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

Dissecting the role of tyrosine phosphorylation of WAVE in macrophage migration

Cell migration is highly dependent on the precise orchestration of the assembly and disassembly of filamentous actin at their leading edge. Therefore, the temporal and spatial regulation of WAVE activity as the main regulator of Apr2/3 for branched actin polymerization is crucial for efficient lamellipodia-driven cell motility. This work shows that wave mutant Drosophila macrophages completely lack lamellipodia that causes severe migration defects. However, they still respond to wound signals by relying on rudimentary filopodia based migration. Furthermore, it could be shown that the disruption of the WIRS receptor binding site within the WRC does not interfere with wound response of macrophages. The non-receptor tyrosine kinase Abl is assumed to be a key regulator of WAVE, altering its activity via phosphorylation. The results in this study confirm that the active form of Abl is an effector of WAVE. The loss of abl and the overexpression of Abl kinase dead transgene leads to an increase of cell spreading. Further, WAVE localization at the membrane is elevated in the absence of Abl. This alteration seems to influence random migration by reducing the explorative behavior of macrophages. However, loss of abl did not affect the responsiveness of macrophages to external damage signals. WAVE possesses four tyrosine residues within the WHD: Y127 is a Src kinase target and Y153 of the Abl kinase. The WAVE phospho-mutant Y153F is still phosphorylated via Abl. This indicates that additional tyrosine sites are targeted by Abl. Nevertheless, the phosphorylation of Y153 results in an elevated F-actin level that indicates the physiological relevance of this tyrosine residue. This suggests that phosphorylation of Y153 activates WAVE and the WRC, which is consistent with the results of previous studies. The analysis of random migrating macrophages revealed that this leads to a reduction of cell speed. In contrast, the phosphorylation of Y127 in the WHD showed neither an impact on the F-actin level nor on the migratory behavior of macrophages. Interestingly, the simultaneous phosphorylation of both residues leads to a drastic reduction of lamellipodial structures in macrophages. These cells exhibited extended filopodia and showed a stellar cell shape comparable to wave mutant macrophages. Consequently, in vivo migrating macrophages show a reduction of cell speed and a negative impact on the persistence of these cells. Individual phospho-mimicking mutation of Y127 or Y153 also do not interfere with the viability of the flies, whereas the simultaneous phosphorylation of both sites lead to late pupal lethality. These results indicate that phosphorylation of multiple tyrosine residues has a negative regulatory effect on WRC function. In conclusion, it can be shown that phosphorylation state of WAVE finetunes WRC activity