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Dynamic analysis of apoptotic cell clearance. Time-lapse recordings of apoptotic cell clearance in a stage 15 embryo. Glia (g), macrophages (m) and ectoderm (e) are labeled with simu-cytGFP (green). Apoptotic cells are marked with the fluorescent Annexin V (red). A. Selected frames from a representative movie are shown. A glial cell engulfing an apoptotic cell is depicted by a rectangle. B. Close up views of the marked rectangle.
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The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditi...
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Context 1
... frames from a movie in which apoptotic cells are labeled with Annexin V, and glia, ectoderm and macrophages are labeled with simu-cytGFP are shown in Figure 2. Each frame is a projection of 3 slices 2 μm each. ...
Context 2
... highlighted one event with a white rectangle where a glial cell is engulfing an apoptotic particle. Note the probing behavior of the glial cell: touching the apoptotic particle a few times without engulfing it and then ending up with engulfment of the Annexin V labeled apoptotic cell (Figure 2). ...
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Citations
... After 1 h washing, secondary antibodies and DAPI were applied at room temperature for 2 h. Primary antibodies used are as follows: chicken anti-GFP (Abcam, 1:1,000), rabbit anti-SIMU 1:100(Shklyar et al, 2013b)), mouse anti-Draper (Developmental Studies Hybridoma Bank, 1:100), rabbit anti-Dcp-1(cell Signaling, 1:100), Mouse anti-mCherry (Invitrogen, 1:1,000). Alexa Fluor 488 and Alexa Fluor 555-conjugated secondary antibodies (Life technologies, 1:100) were used. ...
Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non-professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.
... PI and annexin V are well-characterised reagents used to detect membrane permeabilisation/ necrosis and exposure of PS/apoptosis, respectively [83,84]. Embryos were mounted ventralside up on double-sided sticky tape (Scotch), dehydrated for 7-10 minutes in an air-tight box containing sachets of silica gel (Sigma), then covered in voltalef oil before microinjection. ...
Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage–apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, we have exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Six-microns-under (Simu) leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and retention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player regulating macrophages during resolution of inflammation.
... Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2-3% egg volume of LysoTracker (Molecular Probes) as described in Ref. (33). Recording started 30 min following injection. ...
During Drosophila embryogenesis, a large number of apoptotic cells are efficiently engulfed and degraded by professional phagocytes, macrophages. Phagocytic receptors Six-Microns-Under (SIMU), Draper (Drpr) and Croquemort (Crq) are specifically expressed in embryonic macrophages and required for their phagocytic function. However, how this function is established during development remains unclear. Here we demonstrate that the key regulator of Drosophila embryonic hemocyte differentiation, the transcription factor Serpent (Srp), plays a central role in establishing macrophage phagocytic competence. Srp, a homolog of the mammalian GATA factors, is required and sufficient for the specific expression of SIMU, Drpr and Crq receptors in embryonic macrophages. Moreover, we show that each of these receptors can significantly rescue phagocytosis defects of macrophages in srp mutants, including their distribution in the embryo and engulfment of apoptotic cells. This reveals that the proficiency of macrophages to remove apoptotic cells relies on the expression of SIMU, Crq and/or Drpr. However, Glial Cells Missing (GCM) acting downstream of Srp in the differentiation of hemocytes, is dispensable for their phagocytic function during embryogenesis. Taken together, our study discloses the molecular mechanism underlying the development of macrophages as skilled phagocytes of apoptotic cells.
... Apoptotic cells often undergo cell-surface changes that result in the engulfment by professional or semi-professional phagocytes [1,2]. The exposure of 'eat-me' signals on the apoptotic cell surface promotes the specific recognition and subsequent internalization of the corpse by the phagocytes. ...
Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.
... Image analysis was performed using Zeiss LSM 700 and Imaris (Bitplane) software. Live imaging was carried out by dechorionating embryos, mounting them under Halocarbon oil, injecting 2%-3% egg volume LysoTracker (Molecular Probes) as described in [19]. In situ hybridization was performed using standard procedures. ...
Eiger, the sole Drosophila TNF-alpha homolog, causes ectopic apoptosis through JNK pathway activation. Yet, its role in developmental apoptosis remains unclear. eiger mutant flies are viable and fertile but display compromised elimination of oncogenic cells and extracellular bacteria. Here we show that Eiger, specifically expressed in embryonic neurons and glia, is not involved in developmental neuronal apoptosis or in apoptotic cell clearance. Instead, we provide evidence that Eiger is required for damage-induced apoptosis in the embryonic CNS through regulation of the pro-apoptotic gene hid independently of the JNK pathway. Our study thus reveals a new requirement for Eiger in eliminating damaged cells during development.
Copyright © 2015. Published by Elsevier B.V.
... LysoTracker (Molecular Probes, Leiden, The Netherlands) was injected at 2-3% of the egg volume and mounted in Halocarbon oil, as described. 52 ...
Glial phagocytosis of superfluous neurons and damaged or aberrant neuronal material is crucial for normal development and maintenance of the CNS. However, the molecular mechanisms underlying the relationship between neuronal death and glial phagocytosis are poorly understood. We describe a novel mechanism that is able to synchronize neuronal cell death and glial phagocytosis of dying neurons in the Drosophila embryonic CNS. This mechanism involves c-Jun N-terminal kinase (JNK) signaling, which is required for developmental apoptosis of specific neurons during embryogenesis. We demonstrate that the dJNK pathway gain-of-function in neurons leads to dJNK signaling in glia, which results in upregulation of glial phagocytosis. Importantly, this promotion of phagocytosis is not mediated by upregulation of the glial phagocytic receptors SIMU and DRPR, but by increasing glial capacity to degrade apoptotic particles inside phagosomes. The proposed mechanism may be important for removal of damaged neurons in the developing and mature CNS.
... To monitor the in vivo dynamics of apoptotic cell clearance by (GFP-positive) glial cells, we used an early marker for apoptotic cells, Annexin V, which labels specifically Phosphatidylserine (PS) on apoptotic cell surfaces (van den Eijnde et al., 1998;Shklyar et al., 2013b). In the wild type embryonic CNS most of apoptotic particles are engulfed by glia and co-localize with Annexin V inside the phagocytes (Fig. 6A-A″ and C). ...
... Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2%-3% egg volume Alexa fluor 555 conjugated Annexin V (Molecular Probes) or LysoTracker (Molecular Probes) as described in (Shklyar et al., 2013b). Recording started 30 min following injection. ...
The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.
Apoptotic cell (AC) clearance is a complex process in which phagocytes recognize, engulf, and digest ACs during organismal development and tissue homeostasis. Impaired efferocytosis results in developmental defects and autoimmune diseases. In the current study, we performed RNA-sequencing to systematically identify regulators involved in the phagocytosis of ACs by Drosophila melanogaster macrophage-like S2 cells, followed by targeted RNA interference screening. Wunen2 (Wun2), a homolog of mammalian lipid phosphate phosphatase (LPP), was deemed as required for efferocytosis both in vitro and in vivo. However, efferocytosis was independent of Wun2 phosphatase activity. Proteomic analysis further revealed that Rab11 and its effector Rip11 are interaction partners of Wun2. Therefore, Wun2 collaborates with Rip11 and Rab11 to mediate efficient recycling of the phagocytic receptor βν integrin subunit to the plasma membrane. The loss of Wun2 results in the routing of βv integrin subunit (Itgbn) into lysosomes, leading to its degradation. The deficiency of βv integrin subunit on the cell surface leads to aberrant and disorganized actin cytoskeleton, thereby influencing the formation of macrophage pseudopodia toward ACs and thus failure to engulf them. The findings of this study provide insights that clarify how phagocytes coordinate AC signals and adopt a precise mechanism for the maintenance of engulfment receptors at their cell membrane surface to regulate efferocytosis.
Billions of cells die and are cleared throughout the development and homeostasis of an organism. Either improper death or clearance can lead to serious illnesses. In the adult Drosophila ovary, germline cells can die by programmed cell death (PCD) at three distinct stages; here we focus on cell death that occurs in mid- and late oogenesis. In mid-oogenesis, the germline of egg chambers can undergo apoptosis in response to nutrient deprivation. In late oogenesis, the nurse cells are eliminated through a developmentally regulated, non-apoptotic cell death. In this chapter, we describe several methods to detect cell death and phagocytosis in the Drosophila ovary. DAPI stains the chromatin of all cells and can be used to detect morphological changes in cells that die by different mechanisms. TUNEL labels fragmented DNA, which can occur in both apoptotic and non-apoptotic death. LysoTracker, an acidophilic dye, marks acidic vesicles and some dying cells; therefore, it can be used to study both death and phagocytosis. We also describe several antibodies that can be used to investigate cell death and/or phagocytosis: active caspase Dcp-1, membrane markers, and lamins. Many of these antibodies can be used in combination with GFP fusion transgenes for further analysis; we show Rab5-GFP and Rab7-GFP, which can be used to study phagocytosis in further detail.
