Abstract
Voltage sensitive phosphatases (VSPs), including engineered voltage sensitive PTEN, are excellent tools to rapidly and reversibly alter the phosphoinositide (PI) content of the plasma membrane in vivo and study the tumor suppressor PTEN. However, widespread adoption of these tools is hampered by the requirement for electrophysiological instrumentation to control the activity of VSPs. Additionally, monitoring and quantifying the PI changes in living cells requires sophisticated microscopy equipment and image analysis. Here we present methods that bypass these obstacles. First, we explore technically simple means for activation of VSPs via extracellularly applied agents or light. Secondly, we characterize methods to monitor PI(4,5)P2 and PI(3,4,5)P3 levels using fluorescence microscopy or photometry in conjunction with translocation or FRET based PI probes, respectively. We then demonstrate the application of these techniques by characterizing the effect of known PTEN mutations on its enzymatic activity, analyzing the effect of PTEN inhibitors, and detecting in real time rapid inhibition of protein kinase B following depletion of PI(3,4,5)P3. Thus, we established an approach that does not only allow for rapidly manipulating and monitoring PI(4,5)P2 and PI(3,4,5)P3 levels in a population of cells, but also facilitates the study of PTEN mutants and pharmacological targeting in mammalian cells.
Highlights
Phosphoinositides (PIs) are membrane-bound signaling molecules essential for many cellular processes like migration, proliferation, membrane trafficking, and regulation of ion channels (Balla et al, 2009)
We started by exploring cation channel based strategies to activate the prototypical Ciona intestinalis voltage sensitive phosphatase (VSP) (Ci-VSP), a PI(4,5)P2 5-phosphatase
To compare the performance of the above methods of Ci-VSP activation, we evaluated the maximal response to each stimulus as well as the time required for PI(4,5)P2 depletion and the time required for PI(4,5)P2 recovery after cessation of the stimulus
Summary
Phosphoinositides (PIs) are membrane-bound signaling molecules essential for many cellular processes like migration, proliferation, membrane trafficking, and regulation of ion channels (Balla et al, 2009). Various experimental techniques have been developed to control PI levels in living cells, ranging from pharmacological approaches and genetic manipulations such as over-expression or knock-down of PI-metabolizing enzymes, to more dynamic approaches that allow temporal control over PI levels. Three distinct approaches for the acute manipulation of PI concentrations have been developed, all displaying certain limitations that need to be considered. The FKBP-FRB heterodimerization system recruits a PI metabolizing enzyme (e.g., PI(4,5)P2-5-phosphatase) to the membrane when rapamycin is applied The use of the rapamycin-induced dimerization system is limited to cell types that do not express large amounts of FKBP or FRB protein domains endogenously (Coutinho-Budd et al, 2013) and unwanted effects via endogenous targets of rapamycin have to be considered (Brown et al, 1994)
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