Cells constituting a multicellular organism die in a variety of ways throughout life, and most of them die via apoptosis under normal conditions. The occurrence of apoptosis is especially prevalent during development and in tissues with a high cellular turnover rate, such as the thymus and bone marrow. Interestingly, although the number of apoptotic cells produced daily is known to be innumerable in a healthy adult human body, apoptotic cells are rarely observed. This absence is due to the existence of a cellular process called efferocytosis that efficiently clears apoptotic cells. Studies over the past decades have focused on how phagocytes are able to remove apoptotic cells specifically, swiftly, and continuously, resulting in defined molecular and cellular events. In this review, we will discuss the current understanding of the clearance of apoptotic cells at the molecular level.

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-Mertk^FnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

Phagocytosis of apoptotic cells, called efferocytosis, requires calcium inside and outside of phagocytes. Due to its necessity, calcium flux is sophisticatedly modulated, and the level of intracellular calcium in phagocytes is ultimately elevated during efferocytosis. However, the role of elevated intracellular calcium in efferocytosis remains elusive. Here, we report that Mertk-mediated intracellular calcium elevation is necessary for internalization of apoptotic cells during efferocytosis. Drastic depletion of intracellular calcium abrogated the internalization step of efferocytosis by delaying phagocytic cup extension and closure. Especially, the defect of phagocytic cup closure for internalization of apoptotic cells was caused by impaired F-actin disassembly and the attenuated interaction of Calmodulin with myosin light chain kinase (MLCK), leading to diminished myosin light chain (MLC) phosphorylation. Genetic and pharmacological impairment of the Calmodulin-MLCK-MLC axis or Mertk-mediated calcium influx also resulted in inefficient efferocytosis due to a defect in internalization of the targets. Taken together, our observations imply that intracellular calcium elevation through Mertk-mediated calcium influx facilitates efferocytosis by inducing myosin II-mediated contraction and F-actin disassembly required for internalization of apoptotic cells.

Sang-Ah Lee, Juyeon Lee, Kwanhyeong Kim, Hyunji Moon, Chanhyuk Min, Byeongjin Moon, Deokhwan Kim, Susumin Yang, Hyunjin Park, Gwangrog Lee, Raekil Park, and Daeho Park. Mol. Cells 2021;44:214-22. https://doi.org/10.14348/molcells.2021.2217

Ephexin4 is a guanine nucleotide-exchange factor (GEF) for RhoG and is involved in various RhoG-related cellular processes such as phagocytosis of apoptotic cells and migration of cancer cells. Ephexin4 forms an oligomer via an intermolecular interaction, and its GEF activity is increased in the presence of Elmo, an Ephexin4-interacting protein. However, it is uncertain if and how Ephexin4 is autoinhibited. Here, using an Ephexin4 mutant that abrogated the intermolecular interaction, we report that this interaction impeded binding of RhoG to Ephexin4 and thus inhibited RhoG activation. Mutation of the glutamate residue at position 295, which is a highly conserved residue located in the region of Ephexin4 required for the intermolecular interaction, to alanine (Ephexin4^E295A) disrupted the intermolecular interaction and increased binding of RhoG, resulting in augmented RhoG activation. In addition, phagocytosis of apoptotic cells and formation of membrane ruffles were increased more by expression of Ephexin4^E295A than by expression of wild-type Ephexin4. Taken together, our data suggest that Ephexin4 is autoinhibited through its intermolecular interaction, which impedes binding of RhoG.

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.