Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Summary

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Abstract

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca²⁺ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca²⁺-independent. The atypical PKCs are activated by neither Ca²⁺ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation ^1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail ³. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article ⁴, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

Protocol

1. Preparation and Maintenance of Sf9 Cell Monolayer Cultures

1. Sf9 cell culture and maintenance is performed in a tissue culture hood.
2. Sf9 cells are derived from Spodoptera frugiperda ovarian cells (Sf21 cells) and can be purchased as frozen cells in Grace's media from Invitrogen.
3. Place 8 mL of Grace's insect medium, supplemented (1X), to which 30% fetal bovine serum (FBS) has been added in a 75 cm² cell culture flask with a canted neck.
4. Thaw the frozen tube of Sf9 cells rapidly in a 37°C water bath by moving the tube back and forth (about 1-2 minutes).
5. Transfer Sf9 cells to the 75 cm²

Discussion

We have described a technique to image translocation of fluorescently tagged PKCs in real time in Sf9 cells and in Aplysia sensory neurons. Sf9 cells provide a simple system to image PKC translocation since culturing and transfecting them is pretty straight-forward. In contrast, microinjection of Aplysia sensory neurons takes some time to master; up to a few months. Difficulties related to this technique include electrode clogging. Filtering the injection solution and centrifuging it usually helps but s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sf9 frozen cells	Spodoptera frugiperda ovarian cells	Invitrogen	B825-01
Grace’s Insect Medium, Supplemented (1X), liquid	Reagent	GIBCO, by Life Technologies	11605-094	Use to culture Sf9 cells
Corning 75cm2 canted neck cell culture flask	Tool	Corning	430720	Use to culture Sf9 cells
Fetal Bovine Serum	Reagent	CanSera	CS-C08-500	Supplement Grace’s media with FBS prior to use
Syringe	Tool	BD Biosciences	309604	Use to filter Fast Green solution
Syringe	Tool	BD Biosciences	309602	Use to filter plasmid DNA / Fast Green solution
Filter	Tool	Corning	431229	Use to filter Fast Green solution
Filter	Tool	Corning	431212	Use to filter plasmid DNA / Fast Green solution
Glass Bottom Dish	Tool	MatTek Corp.	P35G-1.5-14-C	Use to culture Sf9 cells prior to transfection and imaging
Cellfectin II Reagent	Reagent	Invitrogen	10362-100	Used to express fluorescently tagged PKCs in Sf9 cells
Fast Green FCF	Reagent	Sigma-Aldrich	F-7252	Use to visualize microinjection of plasmid DNA solution into Aplysia sensory neurons
Thin wall glass capillaries	Tool	World Precision Instruments, Inc.	TW100F-4	Use to make micr–lectrodes for microinjection
Microloader pipette tip	Tool	Eppendorf	CA32950-050	Autoclave before using to fill up the micr–lectrodes for microinjection
PV820 Pneumatic PicoPump	Tool	World Precision Instruments, Inc.	SYS-PV820	Part of microinjection station
Micr–lectrode holder	Tool	World Precision Instruments, Inc.	MPH6S	Use to hold micr–lectrode
Micromanipulator	Tool	Sutter Instrument Co.	MP-85	Use to position micr–lectrode in the three-axis