PKC Translocation

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Introduction

PKC(protein kinase C) is family of protein kinase enzymes which locates downstream of receptor Tyrosine kinase and G-protein coupled receptor, .involved in phosophorylation of various substrates. PKC is involved in wide range of physiological functions, such as cell proliferation, gene expression, receptor regulation, ion channel gating, apoptosis, and the other physiological functions via these substrates and play a central role in intracellular signal transduction. It is known that PKC is activated by phorbol ester typified by PMA (phorbol 12-myristate 13-acetate) and migrates from the cytoplasm to the cell membrane. Here, we introduce the result of imaging and analysis of PKC translocation using PMA treated HeLa cell by using CellVoyager CV8000 and Yokogawa analysis software.

Results

All images were captured by using Yokogawa CV7000 and PKC location changes were analyzed by “Plasma Membrane Translocation” method of analysis software (Fig.1). As a result, PKC particles increased on the cell membrane depending on the PMA concentration, and a PMA dose response curve could be generated from the change in the fluorescent intensity of PKC in the plasma membrane and cytoplasm. (fig2a, 2b). Also, with Yokogawa analysis software, individual particle area and intensity were also generated, so we created a graph based on these data using Spotfire®(Fig.3, Fig.4). In this way, using “Plasma Membrane Translocation” algorithm, we can analyze and describe various protein translocation of nuclear, cytoplasm, and plasma membrane in detail.

Original images acquired using CV8000, and recognized image.

Fig.1 Original images acquired using CV7000, and recognized image.
(Blue: Nuclear Hoechst33342, Green: PKC Alexa Fluor488)
PMA(1000ng/ml) treated wells show PKC translocation to plasma membrane.
In the recognized image, 3μm inner from cytoplasm outline is recognized as plasma membrane area.

Experimental

Experiment
  1. HeLa cells were seeded at 10,000 cells/well at 96 well plate.
  2. PMA treatment for 20min. (final conc : 0~1000ng/ml)
  3. Fixed by formaldehyde.
  4. Immunostain PKC, and stain nuclei with Hoechst33342.
  5. Acquire image with CV7000.
    ・Objective lens : 10X
    ・4 images/ well
    ・Exposure time : Hoechst33342 (405nm) 200 msec Alexa Fluor488 (488nm) 400 msec

Analysis Method

“Plasma Membrane Translocation(Basic Mode)” is used for image analysis. Nucleus recognized by Hoechst33342 staining, cytoplasm and the PKC particle are recognized by Alexa Fluor488 derived fluorescent. Analysis algorithm parameter was modified to detect certain intensity range and area of the object to remove artifact. Plasma membrane was defined as 3um inner from outline of cytoplasm region.

PMA dose response curve

Fig. 2 PMA dose response curve
(a) Total intensity of particle present in the plasm membrane per cell
(b) Intensity ratio (Plasma membrane/Cytosol) of the plasma membrane and cytoplasm

PMA dose response curve

Fig. 3 Total intensity ratio histogram of particle present in the plasma membrane and cytoplasm.
(Plasma membrane/Cytoplasm)

)Images at 0.2 sec. interval extracted from the images of 2.6 sec. after start up to 5.4 sec. Ionomycin was added at 3.0 sec. from the start.(Red-framed image)

Fig. 4 Total intensity ratio of particle at plasma membrane and cytoplasm at each PMA concentration.
(Plasma membrane/Cytosol)Blue color is ≧0.97cell.

Spotfire® is a registered trademark of TIBCO Software Inc.


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