Number and brightness analysis of LRRK2 oligomerization in live cells.
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.
Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
Nicholas G
NG
James
Michelle A
MA
Digman
Enrico
E
Gratton
Barbara
B
Barylko
Xiaodong
X
Ding
Joseph P
JP
Albanesi
Matthew S
MS
Goldberg
David M
DM
Jameson
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases