Phagocytosis and removal of apoptotic cells is an integral part of the cell death program and an important event in remodeling tissue, suppressing inflammation, and regulating the immune response. Defects in this process contribute to human diseases, including inflammatory diseases and autoimmune disorders. Phagocytic clearance of apoptotic cells is a tightly controlled cellular process initiated by exposure of "eat me" flags on the surface of the apoptotic cell. Phagocytic receptors recognize "eat me" signals to trigger downstream signaling cascades, leading to internalization and degradation of cell corpses. To understand how apoptotic cells are properly removed, we perform genetic screens to identify new regulators controlling various aspects of cell corpse clearance, and we take a combined approach involving genetics, cell biology, and biochemistry to study underlying molecular mechanisms in the model organism Caenorhabditis elegans.
Recognition and Engulfment of Apoptotic Cells
In C. elegans, cell corpse engulfment is controlled by more than 10 genes in two partially redundant pathways, the ced-1/6/7 and the ced-2/5/12 pathways, which may converge at ced-10 (RAC1), leading to cytoskeleton rearrangement for engulfment.
We recently identified ttr-52, which encodes a secreted transthyretin-like protein that acts in the ced-1/6/7 pathway to mediate cell corpse engulfment. We found that TTR-52 binds surface-exposed phosphatidylserine (PS), the evolutionarily conserved "eat me" signal, on apoptotic cells and mediates the PS signal through phagocytic receptor CED-1 (Figure 1). Thus, TTR-52 acts as a bridging molecule to mediate recognition of apoptotic cells by cross-linking the PS "eat me" signal with the CED-1 receptor. TTR-52 contains no known PS- or lipid-binding domains; how it binds and transduces the PS signal is thus unclear. We are currently collaborating with Yingfang Liu at the Institute of Biophysics (Beijing, China) to solve the crystal structure of TTR-52 and further study how it transduces the PS "eat me" signal to induce cell corpse engulfment through CED-1.
In addition to TTR-52, we recently identified another secreted lipid-binding protein that acts in the same genetic pathway with TTR-52 to mediate apoptotic cell recognition. Interestingly, these two secreted proteins recognize apoptotic cells in different manners. We are investigating how they coordinate to mediate cell corpse recognition and engulfment.
Exposure of PS on the apoptotic cell surface serves as an evolutionarily conserved "eat me" signal for engulfment. However, how PS is externalized remains poorly understood. Loss of function of wah-1 and scrm-1, the two genes involved in PS externalization, only partially affects cell corpse engulfment, indicating that additional mechanisms are involved. By using PS biosensors expressed in C. elegans, we performed genetic screens to search for mutants with altered or disrupted labeling of cell corpses by PS-binding proteins. Identification and characterization of genes affected in these mutants will help us to understand how the PS "eat me" signal is presented and regulated.
Although previous work has identified many genes required for cell corpse engulfment, the mechanism by which engulfment is inhibited is not well understood. We found that C. elegans myotubularin MTM-1 acts as a negative regulator of cell corpse engulfment. Myotubularin belongs to the large and highly conserved MTM protein family, whose members possess phosphoinositide 3-phosphatase activity. MTM mutations are associated with several human diseases, but their cellular and physiological functions are unknown. We found that MTM-1 negatively regulates cell corpse engulfment through the Rac GTPase CED-10 and its bipartite GEF CED-5/DOCK180-CED-12/ELMO. The negative-regulatory effect of MTM-1 requires both its lipid phosphatase activity and the function of PI3-kinases VPS-34 and PIKI-1. Identifying and characterizing negative regulators will help us understand how cell corpse engulfment is precisely controlled.
How Are Cell Corpses Degraded in Phagocytes?
In contrast to a relative wealth of knowledge on cell corpse engulfment, we know little about the fate of apoptotic cells after phagocytes ingest them. In the past three years, we have identified three new regulators of apoptotic cell clearance that act after engulfment to promote cell corpse degradation by regulating phagosome maturation.
Rab GTPases are master regulators of various membrane trafficking events, but their roles in apoptotic cell clearance were not discovered until recently. We identified two Rab GTPases (UNC-108/RAB2 and RAB-14) and a Rab GTPase activating protein (GAP; TBC-2) required for cell corpse degradation, indicating a crucial role of Rabs in this process. We found that UNC-108, the C. elegans homolog of Rab GTPase 2, and RAB-14 act redundantly to promote cell corpse degradation by controlling phagosomal acidification and phagolysosome formation, whereas TBC-2, a Rab GAP, promotes phagosome maturation by inactivating RAB-5 (Figure 2). Moreover, we found that three Rabs, RAB-14, UNC-108/RAB2, and RAB-7, act in sequence to regulate phagolysosome formation. RAB-14 and UNC-108 recruit lysosomes, whereas RAB-7 mediates fusion of lysosomes with phagosomes. Overexpression of human RAB2 and RAB14 efficiently rescues the persistent cell corpse phenotype of unc-108 and rab-14 mutants, indicating that the regulatory mechanisms are evolutionarily conserved from worms to humans.
Although studies by us and others in the past few years have greatly advanced our understanding of apoptotic cell degradation, more questions remain to be addressed. For instance, four Rabs (RAB-5, RAB-7, RAB-14, and UNC-108/RAB2) have been identified to regulate cell corpse degradation by acting at different steps of phagosome maturation, but how their activities are regulated and how their functions are mediated are poorly understood. Moreover, we know little about the regulation of cellular events during phagosome maturation, including phagosomal acidification and the sequential fusion of phagosomes with early endosomes, late endosomes, and lysosomes. To address these questions, we are taking genetic and biochemical approaches to identify genes involved in (1) regulating Rab function; (2) mediating vesicle budding, docking, and fusion; and (3) controlling specific maturation steps. We will also characterize their functions in apoptotic cell degradation by examining dynamic events occurring during phagosome maturation.
Regulation of Specific Membrane Traffic Events during Phagocytosis and Endocytosis
Apoptotic cell clearance involves extensive remodeling of membranes. The apoptotic cell surface is modified for signal presentation; rearrangements of the cytoskeleton occur in phagocytes for cell corpse engulfment, and maturation of apoptotic cell–containing phagosomes involves dynamic exchange of phagosomal components with various intracellular compartments. We have therefore extended our studies to address questions regarding the regulation of specific membrane traffic events during phagocytosis and endocytosis. From genetic screens to search for genes involved in regulating the PS signal, we identified the P4-ATPase TAT-1 and its chaperone, CHAT-1, a CDC50 family protein. We found that TAT-1 and CHAT-1 localize to plasma membranes and tubular membrane structures along the sorting and recycling pathway (Figure 3). Loss of tat-1 and chat-1 disrupts PS asymmetry across both plasma membranes and endomembranes, thereby affecting cell corpse recognition and various steps of endocytic transport. Our studies indicate that TAT-1 and CHAT-1 maintain membrane PS asymmetry to regulate membrane tubulation required for endocytic sorting and recycling. To further investigate endocytic transport processes that TAT-1 and CHAT-1 regulate, we screened for genes that act together or redundantly with TAT-1, and we are characterizing their functions in endocytic trafficking.
We recently observed that lysosomes are highly dynamic in multiple C. elegans cell types, and loss of this function affects various aspects of animal development. We are using both genetic and cell biological approaches to study how lysosome dynamics is regulated and contributes to metazoan development.
Grants from the Chinese Ministry of Science and Technology provided partial support for these projects.
As of January 17, 2012