Xu et al., (2003). Kinetic analysis of receptor-activated phosphoinositide turnover.
January 2014, model of the month by Christine Seeliger
Original model: BIOMD0000000075
Phosphoinositides are a group of membrane lipids involved as lipid messengers in many signalling pathways. Phosphatidylinositol 4,5-bisphosphate (PIP2), the most frequent double phosphorylated phosphoinositide, is the substrate of phospholipase C (PLC). PLC is activated downstream of many important signaling receptors such as growth factor receptors and catalyses the hydrolysis of PIP2 invoking major downstream signaling events .
Xu et al.  investigate the turnover of PIP2 in N1E-115 neuroblastoma cells in response to Bradykinin stimulation using a combined experimental and modeling approach. N1E-115 cells are a common neuronal cell model that has been used extensively in the past to study voltage gated ion channels and Calcium dynamics .
At first, the cellular phosphoinositide levels were measured experimentally using [3H] showing PI as the major component of the phosphoinositide pool in unstimulated cells. Levels of PIP2 and PIP are considerably lower at around 2%. Based on further estimates the authors derive a membrane density of 4000 molecules. Bradykinin induced PLC activation decreases the overall PIP2 levels within 10s. The recovery of the pools takes about 150s.
The authors build a phospholipid signaling model using the modeling environment VCell (Figure 1). Preliminary versions of this model, for not capable of explaining the amount of IP3 production by the observed decrease in PIP2 levels, the model had to be extended by incorporating stimulated synthesis of PIP2 from PIP and henceforth stimulated synthesis of PIP from PI [BIOMD0000000075].
Furthermore, the model elucidated the need of an initial PIP2 synthesis before its decrease, to account for a slightly later onset of PIP2 depletion in experiments compared to the model. This initial onset of PIP2 synthesis was confirmed experimentally and blocking PI-3 and -4-kinases indicated that those PIP2 synthesising enzymes are the main source of it (Figure 2).
However, the authors reused their model to address the behavior of the reporter PH-GFP. This reporter has a high affinity for PIP2 and IP3 in vitro. However, its is predominantly located at the plasma membrane in unstimulated cells in vivo, indicating its association with PIP2. Activation of PLC causes its relocation to the cytoplasm. Experimental results are currently contradicting as to whether this relocation is mainly a result of the PIP2 or IP3 dynamics.
Figure 2 Experimental and simulated time courses of PIP2 and PIP dynamics after bradykinin induction. The red diamond indicates the initial peak of PIP2 production that let the authors to introduce PIP2 synthesis into their model. Figures are taken from .
Figure 3 Simulated spatial dynamics of PH-GFP concentrations over time in the cytosol (1st row) and at the membrane (2nd row) in comparison with the IP3 (3rd row) and PIP2 (4th row). The initial PIP2 peak at 5 seconds is visible with regards to the PIP2 development but not with PH-GFP recruitment to the membrane. The PH-GFP diffusion barrier introduced by the nucleus is visible at 10-30s in the upper right corner of the cells. Figures are taken from .
Figure 1 Illustration of the implemented PIP2 turnover model in VCell including PH GFP translocation. Reactions displayed in the upper half take place at the membrane while the lower half shows cytosolic reactions. Figure taken from .
Experiments conducted by the authors indicated, that the dynamics of PH-GFP translocation follows the dynamics of PIP2 degradation. However, the time courses did not show the previously observed initial peak of PIP2 production after bradykinin stimulation.
The authors extended their initial model of PIP2 turnover by adding the reporter dynamics and implementing the model using both a compartmental and a realistic two dimensional spatial approach. The latter based on the concrete microscopic geometry of a cell.
The initial PIP2 peak does not manifest in PH-GFP translocation in the simulation either although it is visible with regards to the amount of unbound PIP2 (Figure 3). Another observation validating the spatial model with regards to experiments is the formation of a diffusion barrier for PH-GFP by the nucleus. Reduction of the overall free PIP2 and IP3 amplitude is observed as expected and in line with expectations of PH-GFP sequestering of PIP2 and IP3. Indeed, restricted access of PLC to PIP2 was reported in the original PH-GFP paper by Várnai and Bhalla .
Finally, simulations without phosphoinositide turnover but instantaneous IP3 applications and varying amounts of PH-GFP reporter constructs suggest that conflicting experimental reports as to whether rising IP3 concentrations cause the PH-GFP translocation to the cytoplasm or not could simply be caused by differences in PH-GFP availability. Changes in PH-GFP translocation are higher with lower concentrations of PH-GFP.
The study illustrates how experimental approaches can be guided by additional modeling to gain further and deeper insights into the ongoing processes to derive new testable hypothesis open for further investigation.
- Vanhaesebroeck et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem. (2001);70:535-602.
- Xu et al. Kinetic analysis of receptor-activated phosphoinositide turnover. J Cell Biol. (2003);161(4):779-791.
- Kooper and Adorante. Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange. Am J Physiol Cell Physiol. (2002);282(5):C1000-8.
- Várnai and Balla. Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. J Cell Biol. (1998);143(2):501-510.