Degradation Of Apoptotic Cells Research Articles

The role of secreted proteins in efferocytosis.

The clearance of apoptotic cells known as efferocytosis is the final stage of apoptosis, and includes the recognition, phagocytosis, and degradation of apoptotic cells. The maintenance of tissue homeostasis requires the daily elimination of billions of apoptotic cells from the human body via the process of efferocytosis. Accordingly, aberrations in efferocytosis underlie a growing list of diseases, including atherosclerosis, cancer, and infections. During the initial phase of apoptosis, "Eat-Me" signals are exposed and recognized by phagocytes either directly through phagocyte receptors or indirectly through secreted proteins that function as bridge molecules that cross-link dying cells to phagocytes. Here, we set out to provide a comprehensive review of the molecular mechanisms and biological significance of secreted proteins in apoptotic cell clearance. Specifically, it focuses on how these secreted proteins act as bridging molecules to facilitate the clearance process.

Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy.

Cells and tissues, such as macrophages, express inducible nitric oxide synthase (INOS) after stimulation by certain factors. INOS helps mediate the macrophage inflammatory reaction, but few studies have explored how INOS affects macrophage function in nonalcoholic fatty liver disease (NAFLD). This study investigated the role of INOS-mediated macrophage activity in NAFLD. A high-fat diet was used to establish an NAFLD mouse model. After 12 weeks, blood was collected for immune cell and lipid analyses, and liver tissues were collected for pathological analyses with hematoxylin and eosin and Oil Red O staining. Peritoneal macrophages were extracted in situ, cultured in Dulbecco's modified Eagle's medium, and stimulated with palmitic acid to mimic in vivo conditions for further assays. Real-time polymerase chain reaction, western blot analysis, and immunofluorescence were used to verify the expression of target genes or proteins. In the NAFLD model, INOS expression in macrophages increased, and INOS knockdown significantly decreased the number of macrophages. Pathological examinations confirmed that INOS knockdown slowed NAFLD progression and macrophage infiltration during inflammation. INOS knockdown also enhanced phagocytosis and lipid transport by macrophages, and increased the expression of autophagy-related molecules in macrophages, which improved the autophagy level, promoted apoptotic cell degradation, and maintained intracellular environment homeostasis. These results indicate a correlation between INOS expression and macrophage function in NAFLD.

Methods for Quantitative Efferocytosis Assays.

Efferocytosis, the phagocytic removal of apoptotic cells, is a dynamic process requiring recruitment of numerous regulatory proteins to mediate the uptake, engulfment, and degradation of apoptotic cells. Herein, we describe microscopy-based methods for the enumeration of efferocytic events and characterization of the spatiotemporal dynamics of signaling molecule recruitment during efferocytosis using genetically encoded probes and immunofluorescent labeling. While these methods are illustrated using macrophages, they are applicable to any efferocytic cell type.

Efferocytosis and Its Role in Inflammatory Disorders.

Efferocytosis is the effective clearance of apoptotic cells by professional and non-professional phagocytes. The process is mechanically different from other forms of phagocytosis and involves the localization, binding, internalization, and degradation of apoptotic cells. Defective efferocytosis has been demonstrated to associate with the pathogenesis of various inflammatory disorders. In the current review, we summarize recent findings with regard to efferocytosis networks and discuss the relationship between efferocytosis and different immune cell populations, as well as describe how efferocytosis helps resolve inflammatory response and modulate immune balance. Our knowledge so far about efferocytosis suggests that it may be a useful target in the treatment of numerous inflammatory diseases.

Autophagosomes fuse to phagosomes and facilitate the degradation of apoptotic cells in Caenorhabditis elegans

Autophagosomes are double-membrane intracellular vesicles that degrade protein aggregates, intracellular organelles, and other cellular components. During the development of the nematode Caenorhabditis elegans, many somatic and germ cells undergo apoptosis. These cells are engulfed and degraded by their neighboring cells. We discovered a novel role of autophagosomes in facilitating the degradation of apoptotic cells using a real-time imaging technique. Specifically, the double-membrane autophagosomes in engulfing cells are recruited to the surfaces of phagosomes containing apoptotic cells and subsequently fuse to phagosomes, allowing the inner vesicle to enter the phagosomal lumen. Mutants defective in the production of autophagosomes display significant defects in the degradation of apoptotic cells, demonstrating the importance of autophagosomes to this process. The signaling pathway led by the phagocytic receptor CED-1, the adaptor protein CED-6, and the large GTPase dynamin (DYN-1) promotes the recruitment of autophagosomes to phagosomes. Moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex components. Further observations suggest that autophagosomes provide apoptotic cell-degradation activities in addition to and in parallel of lysosomes. Our findings reveal that, unlike the single-membrane, LC3-associated phagocytosis (LAP) vesicles reported for mammalian phagocytes, the canonical double-membrane autophagosomes facilitate the clearance of C. elegans apoptotic cells. These findings add autophagosomes to the collection of intracellular organelles that contribute to phagosome maturation, identify novel crosstalk between the autophagy and phagosome maturation pathways, and discover the upstream signaling molecules that initiate this crosstalk.

Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.72466.

Abstract 11646: ATVB Outstanding Research Award : WDFY3 is Required for the Efficient Degradation of Engulfed Apoptotic Cells by Macrophages During Efferocytosis

Introduction: Defective clearance of apoptotic cells (ACs) by macrophages (efferocytosis) contributes to unresolved inflammation. Our recent genome-wide CRISPR screen has discovered WDFY3 as a novel regulator required for the efficient uptake of ACs during efferocytosis. WDFY3 is well known as a scaffold protein facilitating the autophagic degradation of aggregated proteins in neurons. Yet, if and how WDFY3 may also be required for the efficient degradation of engulfed ACs by macrophages have not been characterized. Hypothesis: WDFY3 is responsible for the efficient degradation of ACs by interacting with other autophagic proteins to facilitate phagosome-lysosome fusion, a process known as LC3-associated phagocytosis (LAP). Methods: Bone marrow-derived macrophages (BMDMs), peritoneal macrophages (PMs), and mice with myeloid-specific Wdfy3 knockout (LysMCre +/- Wdfy3 fl/fl ) or transgenic overexpression of human WDFY3 ( hWDFY3 Tg ) were used for in vitro and in vivo assays. Results: Wdfy3 knockout BMDMs and PMs showed impaired lysosomal acidification and degradation of engulfed ACs. Mechanistically, Wdfy3 knockout impedes efficient LC3 lipidation upon AC engulfment, the last step required for phagosome-lysosome fusion and subsequent lysosomal degradation. Pull-down assay supports that WDFY3 interacts with GABARAP, but not LC3B, to mediate LC3 lipidation, supporting the direct involvement of WDFY3 protein in the process. At the transcriptomic level, RNA-seq and pathway analyses further revealed that gene sets of ROS/RNS production by phagocytes, fatty acid oxidation, and autophagosome were enriched in differentially expressed genes downregulated in Wdfy3 knockout BMDM, supporting the secondary effects of WDFY3-deficiency on the mRNA expression of genes and pathways essential for cargo degradation. Intriguingly, BMDMs and PMs from hWDFY3 Tg mice showed enhanced AC uptake and degradation both in vitro and in vivo , suggesting that WDFY3 has a wide dynamic range in its regulatory capacity. Conclusion: We discovered a novel role of WDFY3 in the degradation of ACs during macrophage efferocytosis. Enhancing WDFY3 may represent a therapeutic approach to promote the resolution of inflammation in diseases due to defective efferocytosis.

Efferocytosis induces macrophage proliferation to help resolve tissue injury

Apoptotic cell clearance by macrophages (efferocytosis) promotes resolution signaling pathways, which can be triggered by molecules derived from the phagolysosomal degradation of apoptotic cells. We show here that nucleotides derived from the hydrolysis of apoptotic cell DNA by phagolysosomal DNase2a activate a DNA-PKcs-mTORC2/Rictor pathway that increases Myc to promote non-inflammatory macrophage proliferation. Efferocytosis-induced proliferation expands the pool of resolving macrophages invitro and in mice, including zymosan-induced peritonitis, dexamethasone-induced thymocyte apoptosis, and atherosclerosis regression. In the dexamethasone-thymus model, hematopoietic Rictor deletion blocked efferocytosing macrophage proliferation, apoptotic cell clearance, and tissue resolution. In atherosclerosis regression, silencing macrophage Rictor or DNase2a blocked efferocyte proliferation, apoptotic cell clearance, and plaque stabilization. In view of previous work showing that other types of apoptotic cell cargo can promote resolution in individual efferocytosing macrophages, the findings here suggest that signaling-triggered apoptotic cell-derived nucleotides can amplify this benefit by increasing the number of these macrophages.

Phagocyte Chemoattraction Is Induced through the Mcp-1-Ccr2 Axis during Efferocytosis.

Apoptotic cells generated during development and for tissue homeostasis are swiftly and continuously removed by phagocytes via a process called efferocytosis. Efficient efferocytosis can be achieved via transcriptional modulation in phagocytes that have engulfed apoptotic cells. However, such modulation and its effect on efferocytosis are not completely understood. Here, we report that phagocytes are recruited to apoptotic cells being cleared through the Mcp-1–Ccr2 axis, which facilitates clearance of apoptotic cells. We identified Mcp-1 as a modulated transcript using a microarray and found that Mcp-1 secretion was augmented in phagocytes engulfing apoptotic cells. This augmented Mcp-1 secretion was impaired by blocking phagolysosomal degradation of apoptotic cells. Conditioned medium from wild type (WT) phagocytes promoted cell migration, but that from Mcp-1−/− phagocytes did not. In addition, blockade of Ccr2, the receptor for Mcp-1, abrogated cell migration to conditioned medium from phagocytes incubated with apoptotic cells. The intrinsic efferocytosis activity of Mcp-1−/− and Ccr2−/− phagocytes was unaltered, but clearance of apoptotic cells was less efficient in the peritoneum of Mcp-1−/− and Ccr2−/− mice than in that of WT mice because fewer Ccr2-positive phagocytes were recruited. Taken together, our findings demonstrate a mechanism by which not only apoptotic cells but also phagocytes induce chemoattraction to recruit phagocytes to sites where apoptotic cells are cleared for efficient efferocytosis.

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila.

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.

The small GTPase RAB-35 defines a third pathway that is required for the recognition and degradation of apoptotic cells.

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.

Exploding the necroptotic bubble.

The apoptotic death of cells is accompanied by the exposure of “eat-me” signals that serve to prevent necrotic degradation of apoptotic cells, and thereby prevent inflammation, promote resolution of immune responses, and stimulate tissue repair. These “eat-me” signals include the exposure of phosphatidylserine (PS) on the outer plasma membrane during the early stages of apoptosis as well as on the surface of apoptotic bodies, plasma membrane vesicles that are shed during the later stages of cell death. In our recent publication (PLoS Biol. 15(6):e2002711), we describe similar ‘eat-me’ and ‘find-me’ signals present during necroptosis, challenging some of our common assumptions about regulated forms of lytic death.

Phagocytosis Assay for Apoptotic Cells in <em>Drosophila</em> Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Presumptive TRP channel CED-11 promotes cell volume decrease and facilitates degradation of apoptotic cells in Caenorhabditis elegans

Apoptotic cells undergo a series of morphological changes. These changes are dependent on caspase cleavage of downstream targets, but which targets are significant and how they facilitate the death process are not well understood. In Caenorhabditis elegans an increase in the refractility of the dying cell is a hallmark morphological change that is caspase dependent. We identify a presumptive transient receptor potential (TRP) cation channel, CED-11, that acts in the dying cell to promote the increase in apoptotic cell refractility. CED-11 is required for multiple other morphological changes during apoptosis, including an increase in electron density as visualized by electron microscopy and a decrease in cell volume. In ced-11 mutants, the degradation of apoptotic cells is delayed. Mutation of ced-11 does not cause an increase in cell survival but can enhance cell survival in other cell-death mutants, indicating that ced-11 facilitates the death process. In short, ced-11 acts downstream of caspase activation to promote the shrinkage, death, and degradation of apoptotic cells.

GOP-1 promotes apoptotic cell degradation by activating the small GTPase Rab2 in C. elegans.

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.

Decreased serum DNase1-activity in patients with autoimmune liver diseases

Deoxyribonuclease1 (DNase1) is involved in chromatin degradation of apoptotic cells. Its deficiency results in accumulation of self-DNA, which in turn may induce inflammation and autoimmunity. We assessed for the first time serum DNase1-activity in a large consecutive cohort of treatment-naïve patients with autoimmune liver diseases (ALD). DNase1-activity was determined by single radial enzyme-diffusion (SRED) at diagnosis of 224 patients with autoimmune hepatitis (AIH), 249 with primary biliary cirrhosis (PBC) and 36 with primary sclerosing cholangitis (PSC). Sera from 146 patients with chronic hepatitis B or C, 140 with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and 114 healthy individuals served as disease and healthy controls. Available serum samples during remission from 50 AIH and 39 PBC patients were also investigated by paired analyzes. DNase1-activity was significantly lower in AIH, PBC and PSC compared to viral hepatitis (p < 0.02, p < 0.001, p = 0.03), NAFLD/NASH (p < 0.001) and healthy (p < 0.001). No significant difference was found in between each specific ALD. In AIH, DNAse1-activity was positively correlated with aspartate aminotransferase (AST) (p < 0.02), bilirubin (p < 0.01) and increased IgG (>1400 mg/dl; p < 0.05); in PBC, with AST (p < 0.01), alanine aminotransferase (ALT) (p < 0.03) and anti-mitochondrial antibodies (AMA) (p = 0.008). In PSC, DNase1-activity was inversely associated with alkaline phosphatase (ALP) (p < 0.05). In AIH, complete responders were characterized by increased baseline DNase1-activity compared to partial responders, relapsers and non-responders (p < 0.02), whereas it was significantly increased after achievement of remission (p < 0.001). Serum DNase1-activity is significantly decreased in ALD patients, indicating its potential implication in their pathogenesis. Furthermore, DNase1-activity could be used as a new surrogate biomarker for predicting response to AIH treatment.

Microglial Phagocytosis in the Neurodegenerative Diseases

Microglia are the resident innate immune cells in the brain. Under endogenous or exogenous stimulates, they become activated and play an important role in the neurodegenerative diseases. Microglial phagocytosis is a process of receptor-mediated engulfment and degradation of apoptotic cells. In addition, microglia can phagocyte brain-specific cargo, such as myelin debris and abnormal protein aggregation. However, recent studies have shown that microglia can also phagocyte stressed-but-viable neurons, causing loss of neurons in the brain. Thus, whether microglial phagocytosis is beneficial or not in neurodegenerative disease remains controversial. This article reviews microglial phagocytosis related mechanisms and its potential roles in neurodegenerative diseases, with an attempt to provide new insights in the treatment of neurodegenerative diseases.

Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines

Abstract Introduction: Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells. Methods: The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software. Results: In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear. Conclusions: Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle. Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.