FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.

Guanine-nucleotide dissociation inhibitors (GDIs) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor, GDI.Cdc42 FLARE, in which Cdc42 is modified with a fluorescence resonance energy transfer (FRET) 'binding antenna' that selectively reports Cdc42 binding to endogenous GDIs. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed remarkably tight coordination of GTPase release and activation on a time scale of 10 s, suggesting that GDI-Cdc42 interactions are a critical component of the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.

Erk5 activation elicits a vasoprotective endothelial phenotype via induction of Kruppel-like factor 4 (KLF4).

The MEK5/Erk5 MAPK cascade has recently been implicated in the regulation of endothelial integrity and represents a candidate pathway mediating the beneficial effects of laminar flow, a major factor preventing vascular dysfunction and disease. Here we expressed a constitutively active mutant of MEK5 (MEK5D) to study the transcriptional and functional responses to Erk5 activation in human primary endothelial cells. We provide evidence that constitutive Erk5 activation elicits an overall protective phenotype characterized by increased apoptosis resistance and a decreased angiogenic, migratory, and inflammatory potential. This is supported by bioinformatic microarray analysis, which uncovered a statistical overrepresentation of corresponding functional clusters as well as a significant induction of anti-thrombotic, hemostatic, and vasodilatory genes. We identify KLF4 as a novel Erk5 target and demonstrate a critical role of this transcription factor downstream of Erk5. We show that KLF4 expression largely reproduces the protective phenotype in endothelial cells, whereas KLF4 siRNA suppresses expression of various Erk5 targets. Additionally, we show that vasoprotective statins potently induce KLF4 and KLF4-dependent gene expression via activation of Erk5. Our data underscore a major protective function of the MEK5/Erk5/KLF4 module in ECs and implicate agonistic Erk5 activation as potential strategy for treatment of vascular diseases.

MEK5/ERK5 signaling modulates endothelial cell migration and focal contact turnover.

The formation of new blood vessels from pre-existing ones requires highly coordinated restructuring of endothelial cells (EC) and the surrounding extracellular matrix. Directed EC migration is a central step in this process and depends on cellular signaling cascades that initiate and control the structural rearrangements. On the basis of earlier findings that ERK5 deficiency in mouse EC results in massive defects in vessel architecture, we focused on the impact of the MEK5/ERK5 signaling pathway on EC migration. Using a retroviral gene transfer approach, we found that constitutive activation of MEK5/ERK5 signaling strongly inhibits EC migration and results in massive morphological changes. The area covered by spread EC was dramatically enlarged, accompanied by an increase in focal contacts and altered organization of actin filaments. Consequently, cells were more rigid and show reduced motility. This phenotype was most likely based on decreased focal contact turnover caused by reduced expression of p130Cas, a key player in directed cell migration. We demonstrate for the first time that ERK5 signaling not only is involved in EC survival and stress response but also controls migration and morphology of EC.

A mix-and-measure assay for determining the activation status of endogenous Cdc42 in cytokine-stimulated macrophage cell lysates.

Cytokine stimulations of leukocytes many times result in transient activation of the p21 Rho family of small GTPases. The role of these molecules during cell migration and chemotaxis is well established. The traditional approach to study the activation dynamics of these proteins involves affinity pull-downs that are often cumbersome and prone to errors. Here, we describe a reagent and a method of simple "mix-and-measure" approach useful for determining the activation status of endogenous Cdc42 GTPase from cell lysates.

Quantitative ratiometric imaging of FRET-biosensors in living cells.

Biosensors based on FRET have been useful in deciphering the dynamics of protein activation events in living cells at subcellular resolutions and in time scales of seconds. These new systems allow observations of dynamic processes which were not possible previously using more traditional biochemical and cell biological approaches. The image data sets obtained from these sensors require careful processing in order to represent the actual protein activation events. Here, we will cover the basic approaches useful for processing the raw image data sets into relativistic ratiometric measurements, capable of depicting relative differences in the protein activation states within a single cell. We will discuss in detail the approaches for genetically encoded, single-chain biosensor systems based on FRET, as well as those that are based on intermolecular, dual-chain design. Additionally, the same analysis can be utilized for biosensor systems using solvatochromic dyes (Nalbant, Hodgson, Kraynov, Toutchkine, & Hahn, 2004), useful for detection of endogenous protein activation states.

Multiplex imaging of Rho family GTPase activities in living cells.

Here, we provide procedures for imaging the Rho GTPase biosensors in both single and multiplex acquisition modes. The multiplex approach enables the direct visualization of two biosensor readouts from a single living cell. Here, we take as an example a combination of the RhoA biosensor based on a CFP/YFP FRET modality and the Cdc42 biosensor based on organic dyes that change fluorescence as a function of the local solvent polarity. We list the required optical components as well as cellular manipulation techniques necessary to successfully image these two ratiometric biosensors in a single living cell.

Dynamics of the Rho-family small GTPases in actin regulation and motility.

The p21 Rho-family of small GTPases are master regulators of actin cytoskeleton rearrangements. Their functions have been well characterized in terms of their effects toward various actin-modulating protein targets. However, more recent studies have shown that the dynamics of Rho GTPase activities are highly complex and tightly regulated in order to achieve their specific subcellular localization. Furthermore, these localized effects are highly dynamic, often spanning the time-scale of seconds, making the interpretation of traditional biochemical approaches inadequate to fully decipher these rapid mechanisms in vivo. Here, we provide an overview of Rho family GTPase biology, and introduce state-of-the-art approaches to study the dynamics of these important signaling proteins that ultimately coordinate the actin cytoskeleton rearrangements during cell migration.

Sequencing of hemoglobin gene 4 (dmhb4) and Southern blot analysis provide evidence of more than four members of the Daphnia magna globin family.

A genomic fragment containing the hemoglobin gene dmhb4 of Daphnia magna was cloned and its nucleotide sequence determined. Concerning induction under hypoxic conditions, dmhb4 was found to be expressed constitutively with similar mRNA quantities in D. magna bred in either normoxic or hypoxic medium. Southern blot analysis revealed at least six hemoglobin-like sequences in the genome of Daphnia magna.