Efferocytosis requires periphagosomal Ca2+-signaling and TRPM7-mediated electrical activity

Abstract

Efficient clearance of apoptotic cells by phagocytosis, also known as efferocytosis, is fundamental to developmental biology, organ physiology, and immunology. Macrophages use multiple mechanisms to detect and engulf apoptotic cells, but the signaling pathways that regulate the digestion of the apoptotic cell cargo, such as the dynamic Ca2+ signals, are poorly understood. Using an siRNA screen, we identify TRPM7 as a Ca2+-conducting ion channel essential for phagosome maturation during efferocytosis. Trpm7-targeted macrophages fail to fully acidify or digest their phagosomal cargo in the absence of TRPM7. Through perforated patch electrophysiology, we demonstrate that TRPM7 mediates a pH-activated cationic current necessary to sustain phagosomal acidification. Using mice expressing a genetically-encoded Ca2+ sensor, we observe that phagosome maturation requires peri-phagosomal Ca2+-signals dependent on TRPM7. Overall, we reveal TRPM7 as a central regulator of phagosome maturation during macrophage efferocytosis.

PRL-1/2 phosphatases control TRPM7 magnesium-dependent function to regulate cellular bioenergetics

“…TRPM7 patch-clamp recording is mostly limited to the plasma membrane, which might not represent the entire functional activity of this chanzyme as it is mostly localized to intracellular vesicles ( 23 , 24 ). As such, we used our reporter system to characterize potential regulators of TRPM7 overall function.…”

“…It is unclear whether magnesium can be similarly regulated in mammals. Still, in mammalian cells, the CNNMs localized to the plasma membrane and intracellular vesicles similarly to TRPM7 and the PRLs ( 15 , 23 , 24 , 40 , 55 ), suggesting that the interaction might not only be at the plasma membrane. Interestingly, our magnesium reporter described herein can also detect loss of mitochondrial magnesium, indicating that the signal obtained in our assay is not only representative of magnesium flux derived from the extracellular environment.…”

“…Patch-clamp method has been widely considered the gold standard to directly record ion channel activity at the plasma membrane, including TRPM7. Still, TRPM7 is mainly present in intracellular vesicles inaccessible to patch-clamp recording using regular techniques ( 23 , 24 ), indicating that other functions of TRPM7 may have been missed. We believe that the use of the reporter can circumvent this by measuring an overall response following TRPM7 activation.…”

“…Nuclear translocation of the cleaved kinase domain was shown to phosphorylate histones and regulate gene expression ( 22 ). Although TRPM7 is present at the plasma membrane, the majority is located in unique intracellular vesicles ( 23 , 24 ). TRPM7 is involved in cellular proliferation and migration and has been linked to cancer progression ( 25 , 26 ).…”

epHero – a tandem-fluorescent probe to track the fate of apoptotic cells during efferocytosis

“…In recent years, several groups have reported the importance of phagosomal acidification and cargo degradation during efferocytosis [ 28 , 32 ], highlighting the need to successfully measure these processes. One study circumvented the limitations of conventional efferocytosis techniques by combining the pHrodo approach with GFP-expressing apoptotic cells, and employing the ratio of pHrodo to GFP fluorescence to measure acidification [ 28 ]. However, the signal of a fluorescent dye and an intracellular fluorophore can vary greatly.…”