Showing papers by "Klaus M. Hahn published in 2010"

Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

Abstract: Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na+/H+ exchange. Yet, the functional relationship between Na+/H+ exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na+/H+ exchange, elevating cytosolic pH and stimulating Na+ influx. Remarkably, although inhibition of Na+/H+ exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na+ influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na+/H+ exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na+/H+ exchange.

Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo

Abstract: The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in cultured single cells but can either repress or stimulate motility in epithelial cells depending on the conditions. The role of Rac in collective epithelial cell movements in vivo, which are important for both morphogenesis and metastasis, is therefore difficult to predict. Recently, photoactivatable analogues of Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using light. In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in vivo, specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense direction as a group according to relative levels of Rac activity. Communication between cells of the cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies further show that photoactivatable proteins are effective tools in vivo.

Engineered allosteric activation of kinases in living cells

Abstract: Studies of cellular and tissue dynamics benefit greatly from tools that can control protein activity with specificity and precise timing in living systems. Here we describe an approach to confer allosteric regulation specifically on the catalytic activity of protein kinases. A highly conserved portion of the kinase catalytic domain is modified with a small protein insert that inactivates catalytic activity but does not affect other protein functions (Fig. 1a). Catalytic activity is restored by addition of rapamycin or non-immunosuppresive rapamycin analogs. Molecular modeling and mutagenesis indicate that the protein insert reduces activity by increasing the flexibility of the catalytic domain. Drug binding restores activity by increasing rigidity. We demonstrate the approach by specifically activating focal adhesion kinase (FAK) within minutes in living cells and show that FAK is involved in the regulation of membrane dynamics. Successful regulation of Src and p38 by insertion of the rapamycin-responsive element at the same conserved site used in FAK suggests that our strategy will be applicable to other kinases.

Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells

Abstract: The biosensors developed in our laboratory have been based on different designs, each imparting specific strengths and weaknesses. Here we describe detailed protocols for the application of three biosensors exemplifying different designs -- first a design in which an environmentally sensitive dye is used to report the activation of endogenous Cdc42, followed by two biosensors based on FRET, one using intramolecular and the other intermolecular FRET. The design differences lead to the need for different approaches in imaging and image analysis. Figure 1 shows the design of the three biosensors. MeroCBD (Fig 1a) is a sensor for Cdc42 activation in which a fragment of Wiskott Aldrich Syndrome protein that binds only to active Cdc42 is derivatized with an environmentally sensitive dye1. In living cells, the dye undergoes a fluorescence change when the biosensor binds to endogenous, untagged Cdc42. This design is advantageous for several reasons: Cdc42 is not tagged with a fluorophore or other biosensor component, expression of exogenous Cdc42 is not needed, and the signal is substantially brighter than FRET because a bright dye is directly excited, rather than indirectly as in FRET. The major disadvantage is that the biosensor is not genetically encoded so must be loaded in the cells via microinjection, electroporation etc. Fig.1 Fluorescent biosensor designs. A. MeroCBD, biosensor of endogenous Cdc42 activation. Here a fragment of Wiskott Aldrich syndrome protein (WASP) that binds only to activated Cdc42 is covalently derivatized with an environmentally sensitive dye. When the ... The FRET biosensor for RhoA is shown in figure 1B. A fragment of Rhotekin is attached directly to RhoA, and fluorescent proteins undergoing FRET are built into the chain connecting the RhoA and Rhotekin fragment. The C terminus of the RhoA is purposefully left free of fluorescent protein or other modifications, to maintain intact the regulation of RhoA by GDI. Unlike the Cdc42 biosensor, this biosensor is completely genetically encoded, greatly simplifying loading into cells. Finally, the biosensor for Rac1 is shown in Fig.1C. Named Rac1 FLAIR, it is a modification of a design originally published using a covalently attached fluorescent dye for FRET. In the modification described here, the dye has been replaced with a fluorescent protein, rendering the biosensor fully genetically encoded. A fragment of p21 activated kinase (PAK) is used to bind specifically to activated Rac1. The main difference between this design and that of the RhoA biosensor is that the PAK fragment is not attached to Rac1. Rac1 FLAIR is an intermolecular FRET biosensor, frequently referred to as a dual chain sensor. The use of a dual chain design significantly enhances the sensitivity of the biosensor in comparison with single chain FRET biosensors. The latter usually show substantial FRET even when the biosensor is in the `off' state; the two fluorophores are never fully separated. With the Rac1 sensor and other single chain sensors the dynamic range, the difference between the on and off states, is enhanced. There is a downside to the dual chain approach. The two components of the biosensor distribute differently in the cell, necessitating additional steps in image processing. Both single chain and dual chain biosensors are potentially perturbed by competition from native protein ligands; the dual chain sensor may be more likely to produce `false negatives' as it may be more sensitive to ligands that compete for the GTPase binding fragment, while the steric bulk of the single chain sensor may prevent it from reaching all sites normally accessed by the GTPases. The issue of competition and its effect on biosensor readout is complex and beyond the scope of this article. We recently completed a careful study of the effects of varying the concentration of each component of the dual chain Rac1 biosensor. This did not appreciably alter the results of our cell protrusion studies (manuscript submitted).

Endogenous RhoG Is Rapidly Activated after Epidermal Growth Factor Stimulation through Multiple Guanine-Nucleotide Exchange Factors

Abstract: RhoG is a member of the Rac-like subgroup of Rho GTPases and has been linked to a variety of different cellular functions. Nevertheless, many aspects of RhoG upstream and downstream signaling remain unclear; in particular, few extracellular stimuli that modulate RhoG activity have been identified. Here, we describe that stimulation of epithelial cells with epidermal growth factor leads to strong and rapid activation of RhoG. Importantly, this rapid activation was not observed with other growth factors tested. The kinetics of RhoG activation after epidermal growth factor (EGF) stimulation parallel the previously described Rac1 activation. However, we show that both GTPases are activated independently of one another. Kinase inhibition studies indicate that the rapid activation of RhoG and Rac1 after EGF treatment requires the activity of the EGF receptor kinase, but neither phosphatidylinositol 3-kinase nor Src kinases. By using nucleotide-free RhoG pull-down assays and small interfering RNA-mediated knockdown studies, we further show that guanine-nucleotide exchange factors (GEFs) of the Vav family mediate EGF-induced rapid activation of RhoG. In addition, we found that in certain cell types the recently described RhoG GEF PLEKHG6 can also contribute to the rapid activation of RhoG after EGF stimulation. Finally, we present results that show that RhoG has functions in EGF-stimulated cell migration and in regulating EGF receptor internalization.

Hold me tightly LOV

Abstract: Mutations that increase the dynamic range of a photoswitchable protein may be used to improve other such photoswitches and increase their potential for allosteric control of protein activity in live cells.

Genetically encoded photomanipulation of protein and peptide activity

Abstract: The present invention relates to fusion proteins comprising protein light switches and methods of photomanipulating the activity of the proteins. The invention further relates to polynucleotides and vectors encoding the fusion proteins, cells comprising the fusion proteins, and methods of using the fusion proteins to study protein function and analyze subcellular activity, as well as diagnostic and therapeutic methods.