Technical Reference #1709

1709.

CaMKIIβ binding to stable F-actin in vivo regulates F-actin filament stability Yu-Chih Lin and Lori Redmond*, Medical College of Georgia, PNAS, 105(1709), (2008)
Link To Paper

Abstract:
Ca2 /calmodulin-dependent protein kinase II (CaMKII) is a serine/ threonine kinase that is best known for its role in synaptic plasticity and memory. Multiple roles of CaMKII have been identified in the hippocampus yet its role in developing neurons is less well understood. We show here that endogenous CaMKII but not CaMKII localized to prominent F-actin-rich structures at the soma in embryonic cortical neurons. Fluorescence recovery after photobleaching analyses of GFP-CaMKII binding interactions with Factin in this CaMKII -free system indicated CaMKII binding depended upon a putative F-actin binding domain in the variable region of CaMKII . Furthermore CaMKII decreased CaMKII binding to F-actin. We examined the interaction of CaMKII with stable and dynamic actin and show that CaMKII binding to F-actin was dramatically prolonged when F-actin was stabilized. CaMKII binding to stable F-actin was disrupted when it was bound by Ca2 / calmodulin or when it was highly phosphorylated but not by kinase inactivity. Whereas CaMKII over-expression increased the prevalence of the F-actin-rich structures disruption of CaMKII binding to F-actin reduced them. Taken together these data suggest that CaMKII binding to stable F-actin is important for the in vivo maintenance of polymerized F-actin.

Keywords:
cytoskeleton; FRAP

Materials & Methods:
E18 cortical neurons were cultured on 35-mm glass bottom dishes (MatTek) at 0.75 106 cells/dish and imaged at 5 DIV. Before imaging media was replaced with Culture External Base containing 2-mM MgCl2 2-mM CaCl2 150-mM NaCl 2.5-mM KCl 10-mM glucose and 10-mM NaHEPES. Cells were maintained in Culture External Base at room temperature during imaging for a maximum of 2 h. In the FRAP experiments images were taken with 63 objective at 16 zoom. Images were captured every 1 s for 500 s. In Fig. 2 however images were taken every 393.21 ms. A circular ROI for photobleaching was chosen with radius of 0.43 m. Cells were photobleached after 10 frames of imaging with 8 iterations (10 iterations for GFP alone) of maximal excitation power. Background fluorescence Fbkgd was determined in an unbleached area with similar initial fluorescence as the bleached ROI. F(t)ROI was normalized to background fluorescence with an equation of F(t)norm (F(t)ROI/ Fbkgd. The first time-point after the bleach was set to t 0. The normalized fluorescence of the frame immediately before photobleaching F( 1) norm was set as 1. The fluorescence at other time points were normalized to F( 1)norm to generate the final fluorescence value F(t)final F(t)norm/F( 1)norm. Final fluorescence was plotted over time to generate the fluorescence recovery curve. When F(t)final reached 1 it was considered to have completely recovered. The time-point at which fluorescence first reached 1 was determined as the full-recovery time. The half-time of the recovery (t1/2) was determined by the time required to reach 50% of final recovered fluorescence F1/2 (F( ) F (0))/2. The unrecoverable fraction (UF) represents the immobile fraction of molecules in the ROI and was determined by UF (1 F( ))/(1 F (0)). To stabilize actin filaments cells were incubated with 10- g/ml cytochalasin-D for 30 min or 1- M jasplakinolide for 1 h before imaging. Then 10- M KN92 or KN93 were added with jasplakinolide 1 h before imaging. All data were analyzed by Microsoft Office Excel 2003 and Origin 6.1 software. Results are reported as mean SEM. Student’s t test was used to determine statistical significance.

Microscopic Technique
Fluorescence Microscopy, Fluorescence Redistribution After Photobleaching

Cell Type(s)
E18, cortical cultures