No abstract

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-δ1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks1,2. Early clinical trials have shown promise, especially in acute myeloid leukaemia3, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.