Kinetic properties of ASC protein aggregation in epithelial cells

Abstract

Apoptosis-associated speck-like protein with CARD domain (ASC), an adaptor protein composed of caspase recruitment and pyrin domains, can efficiently self-associate to form a large spherical structure, called a speck. Although ASC aggregation is generally involved with both inflammatory processes and apoptosis, the detailed dynamics of speck formation have not been characterized. In this report, speck formation in HeLa cells transfected with ASC is examined by time-lapse live-imaging by confocal laser scanning microscopy. The results show that ASC aggregation is a very rapid and tightly regulated process. Prior to speck formation, soluble ASC aggregation is a low probability event, and the affinity of ASC subunits for one another is very low. Following a speck nucleation event, the affinity for further addition of ASC subunits increases dramatically, and aggregation is a highly energetically favorable reaction (Gibbs free energy approximately -40 kJ/mol). This leads to a rapid depletion of soluble ASC, making it highly unlikely that a second speck will form inside the same cell and assuring that speck formation is "all or none," with a well-defined end point. Comparison with kinetic models of the aggregation process indicates diffusion, instead of active transport, is the dominant process for speck growth. Though speck formation and aggresome formation share some properties, we show that the two processes are distinct.

A Novel Flow Cytometric Method To Assess Inflammasome Formation

“…Upon treatment with inflammasome stimuli such as the NLRP3 agonist nigericin, a potassium ionophore, ASC rapidly relocates to form the inflammasome speck. This appears to be an "all or nothing" response, with most of the ASC in the cell accumulating in the speck within minutes of initiation (12). We have taken advantage of this rapid and striking relocalization of ASC to develop a flow cytometric assay for inflammasome activity.…”

Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

“…For some NLRs, such as NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) mediates inflammasome assembly by interacting with capase-1 and the NLR (11,13,15). Complexes of NLRs and ASC form large structures in macrophages that can be detected as foci by microscopic techniques (17)(18)(19). In cases where inflammasome assembly is …”

“…For some NLRs, such as NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) mediates inflammasome assembly by interacting with capase-1 and the NLR (11,13,15). Complexes of NLRs and ASC form large structures in macrophages that can be detected as foci by microscopic techniques (17)(18)(19). In cases where inflammasome assembly is triggered in a T3SS-dependent manner upon infection of macrophages with pathogens, specific PAMPs identified to activate caspase-1 upon contaminating the cytosol include ectopically translocated flagellin, rod proteins, needle proteins, and translocated effector proteins (2,(11)(12)(13)15).…”

NLRC4 inflammasomopathies

“…Upon activation, the inflammasome rapidly forms a large wheel-shaped structure (5), exhausting cellular ASC stores. Inflammasome formation initiates pyroptosis, a form of inflammatory cell death (6) and also proteolytically activates pro-interleukin 1 family cytokines (IL-1β and IL-18) into their cleaved, active forms (7–9).…”