Immunogold electron microscopic demonstration of distinct submembranous localization of the activated gammaPKC depending on the stimulation

Abstract

We examined the precise intracellular translocation of gamma subtype of protein kinase C (gammaPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged gammaPKC (gammaPKC-GFP) on the plasma membrane. In contrast, treatment with Ca(2+) ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of gammaPKC-GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that gammaPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of gammaPKC-GFP on the plasma membrane, Ca(2+) ionophore and NMDA rapidly translocated gammaPKC-GFP to the plasma membrane and then restricted gammaPKC-GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca(2+) influx alone induced the association of gammaPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of gammaPKC in response to various stimuli suggests a novel mechanism for PKC activation.

Figure 1

Quantitative analysis of PKC localization. (A) Visualization of the gap between top and bottom surface of the section. Thickness of ultrathin section (60 nm) resulted in localization of gold particles beneath the plasma membrane or along the outer edge of the plasma membrane when antibody was applied to the top or bottom surface, respectively. (B) Left: subcellular distribution of γPKC–GFP as determined by gold particles. A rectangular area (0.5 × 2.5 μm) placed vertical to and attached to the plasma membrane was selected as shown in the model. Each rectangle was divided into five squares (0.5 × 0.5 μm). The square was further derived into five rectangular areas (0.1 × 0.5 μm). Gold particles in each square were counted and results were expressed as percentage of total counts in each rectangle. Right: submembrane distribution of γPKC–GFP as determined by gold particles. Step 1: The areas were selected within 200 nm from the cell margin. Step 2: Twenty gold particles were counted and each adjacent gold particle was joined. Measurement of the joined lines was calculated together. Length of the plasma membrane was defined to respond to the distribution of the 20 gold particles. Results were expressed as ratio of the measurement/length.

Figure 2

Plasma membrane translocation of γPKC–GFP induced by TPA, A23187, UTP, or NMDA was followed by confocal laser-scanning microscopy. TPA treatment induced the slow and irreversible translocation of γPKC–GFP from the cytoplasm to the plasma membrane (top row). A23187 treatment induced the rapid and reversible translocation from the cytoplasm to the plasma membrane (second row). UTP induced the rapid and reversible translocation (third row). NMDA treatment to the CHO-K1 cells coexpressing NMDA receptors induced the rapid and reversible translocation of γPKC–GFP from the cytoplasm to the plasma membrane (fourth row). Bar = 10 μm.

Figure 3

Immunoelectron microscopic localization of γPKC–GFP in resting CHO-K1 cells. γPKC–GFP immunoreactivity was visualized with immunogold anti-GFP antibodies. Gold particles were distributed throughout the cytoplasm and nucleus and not associated with specific organelles. Two cell samples are shown. Bar = 200 nm.

Figure 4

Immunoelectron microscopic localization of γPKC–GFP in response to TPA. γPKC–GFP immunoreactivity 10 min after TPA stimulation (1 μM) was observed on the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with top side (top), bottom side (bottom), and both sides (both). Two cell samples are shown. Bar = 200 nm.

Figure 5

(A,B) Immunoelectron microscopic localization of γPKC–GFP after A23187 treatment. γPKC–GFP immunoreactivity 10 sec after A23187 stimulation (5 μM) was observed onto the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with both sides. Two cell samples are shown. (C,D) Immunoelectron microscopic localization of γPKC–GFP after UTP treatment. γPKC–GFP immunoreactivity 10 sec after UTP treatment (1 mM) was observed on the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with bottom side. (E,F) Immunoelectron microscopic localization of γPKC–GFP in CHO-K1 cells coexpressing NMDA receptors after NMDA treatment. γPKC–GFP immunoreactivity 10 sec after NMDA treatment (1 mM) was observed onto the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with both sides. Bar: A,B,E,F = 200 nm; C,D = 500 nm.

Figure 6

Immunoelectron microscopic localization of γPKC–GFP after A23187 treatment. γPKC–GFP immunoreactivity 20 sec after A23187 stimulation (5 μM) was observed on the plasma membrane and also just beneath the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with top side or both sides. Two cell samples are shown. Bar = 200 nm.

Figure 7

Quantification of γPKC–GFP immunoreactivity in the 2.5-μm (A) or 0.5-μm (B) region from the plasma membrane. Cells were stimulated with TPA (1 μM, 10 min) and A23187 stimulation (5 μM, 10 sec, or 20 sec). Results are expressed as percentage ± SEM of the total number of gold particles in the 2.5-μm rectangle (n=5). *p<0.01 vs the distribution of γPKC–GFP immunoreactivity without stimulation.

Figure 8

(A,B) Immunoelectron microscopic localization of γPKC–GFP after UTP treatment. γPKC–GFP immunoreactivity 20 sec after UTP treatment (1 mM) was observed on the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with bottom side. Two cell samples are shown. (C,D) Immunoelectron microscopic localization of γPKC–GFP in CHO-K1 cells coexpressing NMDA receptors after NMDA treatment. γPKC–GFP immunoreactivity 20 sec after NMDA treatment (1 mM) was observed on the plasma membrane and also just beneath the plasma membrane. Grids with ultrathin sections were developed with the solution of antibody with bottom side. Two cell samples are shown. Bar = 200 nm.

Figure 9

Quantification of γPKC–GFP immunoreactivity in the 200-nm region from the plasma membrane. Cells were stimulated with TPA (1 μM, 10 min), A23187 (5 μM, 10 sec, and 20 sec, respectively), UTP (1 mM, 10 sec and 20 sec, respectively), and NMDA (1 mM, 10 sec, and 20 sec, respectively). Results are expressed as a ratio ± SEM of the total number of measurement/length (Figure 1B; Step 2) (n=5). Each ratio was compared by best-subset selection procedure. *p<0.01 vs the ratio at TPA stimulation.