E-Poster with pre-recorded video presentation Lorne Infection and Immunity 2021

Live imaging of pyroptotic macrophages reveals novel GSDMD-dependent cytoskeletal remodelling (#261)

Caroline L Holley 1 , Mercedes Monteleone 1 , Kate Schroder 1
  1. University of Queensland, Kangaroo Point, QLD, Australia

The NLRP3 inflammasome assembles in response to a wide range of microbial and host stress signals that are indicative of infection or cell damage. It is well-characterised that upon activation, NLRP3 drives the redistribution of ASC into a large cytosolic cluster, known as the ‘speck’. Inactive caspase-1 undergoes proximity-induced dimerisation and self-cleavage at the speck to generate a transiently active protease. Active caspase-1 triggers a form of inflammatory cell death (pyroptosis) by cleaving full-length GSDMD, releasing a p30 fragment which can oligomerise to perforate the plasma membrane. Loss of plasma membrane integrity causes membrane rupture and release of cytosolic contents into the extracellular space. Cytoskeleton dynamics during NLRP3 inflammasome signalling are not well characterised, and our data show that cytoskeletal catastrophe is an underappreciated yet important feature of pyroptosis. We use high resolution live microscopy, including lattice light sheet microscopy, to characterise these cytoskeletal events in pyroptotic primary murine macrophages. Here, we show that macrophages assemble caspase-1-dependent stress-fibres upon treatment with NLRP3 activating-stimuli. Though much of this cellular  polymerised actin (F-actin) is depolymerised and released upon pyroptotic cell lysis, we discovered that macrophages rapidly assemble filopodia at the plasma membrane upon GSDMD pore assembly. Extracellular F-actin is linked to a number of pathologies, including septic shock, and so F-actin production during NLRP3-dependent pyroptotic cell death may represent a novel pro-inflammatory pathway.