Abstract
ABSTRACTEndocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.
Highlights
Plasma membrane integrity is critical for cellular homeostasis, and wounds in the plasma membrane need to be sealed rapidly to avoid cell death
Endocytic proteins influence the efficiency of membrane repair following LLO-induced plasma membrane damage To study membrane repair following damage from the pore-forming toxins (PFTs), LLO, we made use of a propidium iodide (PI) assay to monitor the permeability of the plasma membrane
Endocytosis has been suggested to play an important role during membrane repair of PFT pores as well as other types of membrane damage
Summary
Plasma membrane integrity is critical for cellular homeostasis, and wounds in the plasma membrane need to be sealed rapidly to avoid cell death For this purpose, cells have developed elaborate membrane repair mechanisms that are triggered by the influx of extracellular Ca2+ into the cytosol. The mechanism for how the plasma membrane is repaired is contested and the current models include (i) patching using intracellular vesicles, (ii) microvesicle shedding, and (iii). Pore-forming toxins (PFTs) that belong to the group of cholesterol-dependent cytolysins form large stable protein-lined pores in the plasma membrane. To repair this type of damage, the protein pore needs to be removed from the cellular membrane. PFT-induced plasma membrane damage is believed to be repaired either via shedding of pore-containing microvesicles or by endocytosis of the pore (Andrews et al, 2014)
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