CaMKIIbeta binding to stable F-actin in vivo regulates F-actin filament stability

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 CaMKIIbeta, but not CaMKIIalpha, localized to prominent F-actin-rich structures at the soma in embryonic cortical neurons. Fluorescence recovery after photobleaching analyses of GFP-CaMKIIbeta binding interactions with F-actin in this CaMKIIalpha-free system indicated CaMKIIbeta binding depended upon a putative F-actin binding domain in the variable region of CaMKIIbeta. Furthermore, CaMKIIalpha decreased CaMKIIbeta binding to F-actin. We examined the interaction of CaMKIIbeta with stable and dynamic actin and show that CaMKIIbeta binding to F-actin was dramatically prolonged when F-actin was stabilized. CaMKIIbeta 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 CaMKIIbeta over-expression increased the prevalence of the F-actin-rich structures, disruption of CaMKIIbeta binding to F-actin reduced them. Taken together, these data suggest that CaMKIIbeta binding to stable F-actin is important for the in vivo maintenance of polymerized F-actin.

Fig. 1.

CaMKIIβ preferentially localized to F-actin rich structures. (A) Confocal images of an E18 cortical neuron immunostained at 4 DIV with antibodies specific for CaMKIIβ (red) and F-actin labeled with fluorescent conjugated phalloidin (green). Colocalization can be seen as yellow in the merged image. (B) Confocal images of cortical neurons that were immunostained at 4 DIV with antibodies that recognize CaMKIIα, δ, and γ isoforms (green). F-actin was labeled with fluorescent conjugated phalloidin (red). (C) Western blot analysis of cortices from rats at embryonic day 18 (E18), postnatal day 3 (P3), P7 and adult with CaMKIIα and CaMKIIβ specific antibodies. E18 4 DIV is homogenate of E18 cortical cultures at 4 days in vitro. Shown on the right are shorter exposures of P7 and adult cortex samples. (D) E18 cortical cultures were harvested at the DIV indicated and Western blot analyses performed with CaMKIIα and CaMKIIβ-specific antibodies. GAPDH is shown as a loading control. (E) Confocal images of an E18 cortical neuron at 4 DIV that was transfected with GFP-CaMKIIβ and labeled with GFP antibodies (green) and fluorescent conjugated phalloidin (red). (F) Confocal images of an E18 cortical neuron at 4 DIV that was transfected with GFP-CaMKIIα and immunostained with CaMKIIβ (red) and GFP (green) antibodies. (G) Confocal images of an E18 cortical neuron at 4DIV that was transfected with GFP-CaMKIIβ-355–393 and labeled with GFP antibodies (green) and fluorescent conjugated phalloidin (red). (H) Enrichment (ratio of microspike/soma mean intensity) of GFP-tagged proteins as indicated in microspikes. **, P < 0.01; *, P < 0.05 compared with CaMKIIβ. (Scale bars, 5 μm.)

Fig. 2.

CaMKIIβ bound to F-actin in the F-actin rich structures. (A) Confocal images of a GFP-CaMKIIβ transfected neuron before bleaching (−0.06 s), at bleach time 0 s, and 5 s after bleaching are shown as examples. GFP-CaMKIIβ enriched areas can be seen as white pixels. A region of interest (ROI) (circle 1) to be bleached was chosen from GFP-CaMKIIβ fluorescently intense areas. (Scale bar, 1 μm.) (B) Fluorescence recovery of GFP, GFP-CaMKIIβ, and paraformaldehyde cross-linked GFP-CaMKIIβ. Fluorescence intensity in the ROI was normalized to an unbleached ROI. (C) Fluorescence recovery of GFP-CaMKIIβ, GFP-CaMKIIα, and GFP-CaMKIIβ-Δ355–392. (D) Time in which fluorescence recovery of GFP-CaMKIIβ, GFP-CaMKIIα, and GFP-CaMKIIβ-Δ355–392 reached half-maximal recovery and full recovery. *, P < 0.05 compared with CaMKIIβ. (E) CaMKIIα expression increased CaMKIIβ recovery rate. Fluorescence recovery of GFP-CaMKIIβ when cotransfected with either control vector, CaMKIIα at 1:1 molar ratio, or at 1:2 molar ratio is shown. (F) Time in which fluorescence recovery of GFP-CaMKIIβ cotransfected with either control vector, CaMKIIα at 1:1 molar ratio, or at 1:2 molar ratio reached half maximal recovery. *, P < 0.05. (G) The percentage of cells transfected as indicated that contained F-actin-rich microspikes. GFP-CaMKIIβ was cotransfected with CaMKIIα at 1:1 molar ratio. Data for each condition is from a minimum of 350 cells from two independent experiments. *, P < 0.05.

Fig. 3.

CaMKIIβ bound stable F-actin stronger than dynamic F-actin. (A and B) Fluorescence recovery of GFP-CaMKIIβ (A) and GFP-CaMKIIα (B) in 1-μM jasplakinolide (JS), 10-μg/ml cytochalasin D (CD), or untreated (control) conditions. CaMKIIβ recovered by 100 s in control conditions, but not when actin was stabilized. (C) Half-maximal fluorescence recovery times for GFP-CaMKIIβ (open bar) and GFP-CaMKIIα (stripes) after either untreated, jasplakinolide, or cytochalasin D treatment. The time to half-maximal recovery was significantly increased in conditions where F-actin was stabilized. (D) The fraction of CaMKIIβ and CaMKIIα that did not recover by 100 s are shown as unrecoverable fractions. In (C) and (D): *, P < 0.05; **, P < 0.01 (difference from control); #, P < 0.05 (CaMKIIβ versus CaMKIIα).

Fig. 4.

Ca2⁺/Calmodulin, but not kinase activity, altered CaMKIIβ binding to F-actin. (A) Disruption of Ca2⁺/CaM binding decreased CaMKIIβ recovery from stable F-actin. Fluorescence recovery of GFP-CaMKIIβ after jasplakinolide and either 10-μM KN93, 10-μM KN92, or control treatment. (B) Half-maximal fluorescence recovery time and the unrecoverable fraction of GFP-CaMKIIβ after treatments as indicated. *, P < 0.05. The unrecoverable fraction of KN93 treatment approached significance from control, P = 0.059. (C) Fluorescence recovery of GFP-CaMKIIβ, GFP-CaMKIIβ-A303R, and GFP-CaMKIIβ-K43R. (D) Half-maximal fluorescence recovery times and the unrecoverable fraction for GFP-CaMKIIβ, GFP-CaMKIIβ-A303R, and GFP-CaMKIIβ-K43R.

Fig. 5.

Activated CaMKIIβ bound less with F-actin resulting in fewer microspikes. (A) Confocal images of an E18 cortical neuron labeled with fluorescent conjugated phalloidin (red) and immunostained with phospho-CaMKII specific antibodies (green). Colocalization can be seen as yellow in the merged image. (Scale bars, 5 μm.) (B) Enrichment of endogenous CaMKIIβ and phospho-CaMKII in microspikes under basal culture conditions show strong colocalization in microspikes. R = 0.90085, P < 0.0001. (C) CaMKIIβ immunoprecipitates of cortical cultures that were either unstimulated (control) or stimulated with 50-mM KCl for 20 min. Western blots of immunoprecipitates were probed with phospho-CaMKII and CaMKIIβ-specific antibodies. KCl stimulation increased CaMKIIβ phosphorylation 1.4018-fold. (D) Confocal images of an E18 cortical neuron stimulated with 50-mM KCl for 20 min and labeled with CaMKIIβ-specific antibodies (red) and fluorescent conjugated phalloidin (green). (E) Confocal images of an E18 cortical neuron transfected with GFP-CaMKIIβ-T287D, fixed, and labeled with fluorescent conjugated phalloidin (red) and immunostained with GFP antibodies (green). (F) Fluorescence recovery of GFP-CaMKIIβ and GFP-CaMKIIβ-T287D after jasplakinolide treatment. (G) Half-maximal and the unrecoverable fraction for GFP-CaMKIIβ and GFP-CaMKIIβ-T287D after jasplakinolide treatment. GFP-CaMKIIβ-T287D t_1/2 recovery was significantly faster. *, P < 0.05. (H) The percentage of cells transfected with GFP, GFP-CaMKIIβ, GFP-CaMKIIβ-T287D (βT287D), GFP-CaMKIIβ-A303R (βA303R), and GFP-CaMKIIβ-K43R (βK43R) that contained F-actin rich microspikes. Data for each condition are from a minimum of 50 cells from three independent experiments. *, P < 0.05. (I) The percentage of cells that contained F-actin rich microspikes in untreated (control) or after 20 min treatment with 50-mM NaCl or 50-mM KCl. Data for each condition are from a minimum of 480 cells from four independent experiments. *, P < 0.05.