Kinetic properties of ASC protein aggregation in epithelial cells.
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.
Cheng
Jun
J
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109-2099, USA.
Waite
Andrea L
AL
Tkaczyk
Eric R
ER
Ke
Kevin
K
Richards
Neil
N
Hunt
Alan J
AJ
Gumucio
Deborah L
DL
D001693:Biological Transport, Active; D053468:CARD Signaling Adaptor Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003198:Computer Simulation; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004058:Diffusion; D004847:Epithelial Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D008954:Models, Biological; D055503:Protein Multimerization; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; C402202:PYCARD protein, human; D011993:Recombinant Fusion Proteins