Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components

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

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

Persistent and polarised global actin flow is essential for directionality during cell migration

Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence

Cells on film - The past and future of cinemicroscopy

ABSTRACT Movie making is now a ubiquitous experimental tool that biologists use alongside more traditional techniques such as molecular biology and biochemistry. It is no longer just cell biologists, but scientists from many other disciplines, such as immunology and neuroscience, that utilise movies to dissect their processes of interest. When did filming become such a standard laboratory technique? Who developed the use of the movie as an experimental tool? The Wellcome Library has recently restored and digitized a number of original 16-mm films from two pioneering cinemicroscopists, Ronald Canti and Michael Abercrombie, which are now freely available to the scientific community. In light of these films, this Essay will give a brief history of the early cinemicroscopists and discuss what is driving the use of movies in the laboratory today.</jats:p

Derivation and simulation of a computational model of active cell populations:How overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces affect alignment

Collective alignment of cell populations is a commonly observed phenomena in biology. An important example are aligning fibroblasts in healthy or scar tissue. In this work we derive and simulate a mechanistic agent-based model of the collective behaviour of actively moving and interacting cells, with a focus on understanding collective alignment. The derivation strategy is based on energy minimisation. The model ingredients are motivated by data on the behaviour of different populations of aligning fibroblasts and include: Self-propulsion, overlap avoidance, deformability, cell-cell junctions and cytoskeletal forces. We find that there is an optimal ratio of self-propulsion speed and overlap avoidance that maximises collective alignment. Further we find that deformability aids alignment, and that cell-cell junctions by themselves hinder alignment. However, if cytoskeletal forces are transmitted via cell-cell junctions we observe strong collective alignment over large spatial scales

A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components