engleski

Rac1 dynamics in Dictyostelium cells

Dictyostelium cells are capable of completely reversing their polarity within 20 seconds, and thus undergo the fastest spontaneous re-polarization among eukaryotic cells. Signalling by small Rho GTPases plays an essential role in this process. Biosensors are therefore needed that are able to match these fast dynamics and enable protracted imaging at high recording rates. However, standard tools used to investigate Rho GTPase activity in living cells such as probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and photoactivation, are of limited usefulness in quickly migrating cells because short time available for image acquisition often results in an unacceptably low signal-tonoise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. We describe characterization of a new fluorescent probe that selectively binds to active forms of Dictyostelium discoideum Rac1 GTPases, and demonstrate its excellent properties for live cell imaging. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase, and was selected on the basis of yeast two-hybrid screen, GST pulldown assay, and FRET measurements by fluorescence lifetime imaging microscopy (FLIM). The probe binds specifically to active Rac1 and has a low cytoplasmic background, thus enabling quantitative determination of the Rac1 activity in the cell membrane over an order of magnitude. The main advantage of DPAKa(GBD) in comparison to similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables measurements of the Rac1 activity in different parts of the membrane. Overexpression of DPAKa(GBD)-DYFP probe induces no adverse effects on cell motility, cytokinesis and growth, thus enabling long-term imaging with negligible photobleaching and phototoxicity. Rac1 GTPases play a dual role in the regulation of actin assembly, which is a key determinant of cell polarity. At the cell front, Rac1 activates the Scar/WAVE complex and thereby stimulates the Arp2/3-mediated actin polymerization. At the cell back, Rac1 regulates stability of the cell cortex by initiating formation of a complex containing IQGAP-related protein DGAP1 and a heterodimer of actin-bundling proteins cortexillins. We will discuss the possible consequences of the Rac1 interaction with multiple partners on its dynamics in living motile cells. Additionally, we will propose that DGAP1 unifies the roles of an effector and a sequestrator in relation to Rac1. Unlike classical effectors, which bind their activators transiently leading to short- lived signalling complexes, interaction between DGAP1 and Rac1-GTP could be stabilized by the formation of a complex with cortexillins. Since the oppositely localized Rac1 effector, Scar/WAVE, promotes actin polymerization at the cell front, competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. This hypothesis is further being investigated by combining imaging and modelling approaches.

Rac1 ; FRET ; small GTPases ; fluorescent probe ; cell polarity

nije evidentirano