Biomedical subjects

Harriët M Loovers

Publications and source records attributed to Harriët M Loovers.

4 recordsLinked to original sources

PubMed2006

Importance of the Hsp70 ATPase domain in yeast prion propagation.

The Saccharomyces cerevisiae non-Mendelian genetic element [PSI+] is the prion form of the translation termination factor Sup35p. The ability of [PSI+] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PSI+]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70-Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PSI+] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties.

Adenosine Triphosphatases↗

PubMed2006

Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase.

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.

Actins↗

PubMed2004

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate [PtdIns(3,4,5)P(3)]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PH(Crac)-GFP from the cytosol to multiple patches, which are similar to the single patch of PH(Crac)-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP) but not the size, lifetime or intensity of patches, indicating that patches are self-organizing structures. A pseudopod is extended from the area of the cell with a PH(Crac)-GFP patch at about 10 seconds after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localized patches of PH(Crac)-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PH(Crac)-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PH(Crac)-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.

Actins↗

PubMed2004

Chemotaxis: signalling modules join hands at front and tail.

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.

Animals↗