Chapter 21: Quantitative fluorescence lifetime imaging in cells as a tool to design computational models of ran-regulated reaction networks.
The understanding of cell function often requires that complex intracellular pathways are considered in quantitative terms. The access to precise measurement of activities in live cells is increasingly provided by the advances in fluorescence lifetime imaging microscopy (FLIM) of Förster (or fluorescence) resonance energy transfer (FRET)-based molecular biosensors. We discuss how quantitative in vivo imaging can be combined with in vitro biochemical characterization to develop computational systems models simulating the behavior of biochemical networks. We describe the application of such approach in computational modeling of the Ran GTPase function in mitotic spindle assembly. The RanGTP concentration gradient surrounding mitotic chromosomes induces the formation of a gradient of spindle assembly factors (SAFs) activated by RanGTP-induced release from inhibitory complexes of SAFs with importin beta. To visualize the gradient of activated SAFs in live cells, we used FLIM to detect the signal of a FRET-based biosensor that reports its RanGTP-induced liberation from importin beta. We review the technical aspects of FRET measurements by FLIM and discuss the step-wise design of systems models aided by quantitative imaging. As evidenced by the example of mitotic Ran gradient studies, the computational simulations are a powerful tool to test new hypotheses on biological function.
Kalab
Petr
P
Department of Cell Biology, University of California, Berkeley, California 94720, USA.
Pralle
Arnd
A
D015374:Biosensing Techniques; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008941:Spindle Apparatus / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D028961:beta Karyopherins; D020931:ran GTP-Binding Protein