Ustilago maydisMating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bölker, M., and Kahmann, R. 1996.Ustilago maydismating hyphae orient their growth toward pheromone sources.Fungal Genetics and Biology20,299–312. When small drops ofUstilago maydissporidia were placed 100–200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example,a2b2mating hyphae grew towarda1b1anda1b2mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with thea2allele began elongating before sporidia with thea1allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous atbbut not ataformed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, thea2a2b1b2strain formed filaments more quickly than thea1a1b1b2strain. Taken together, these results suggest that thea2pheromone diffuses less readily or is degraded more quickly than thea1pheromone.     

Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered,Phospholipase D (PLD)-mediated Cancer Cell Invasion

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of four microRNAs (miRs) mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitant with high expression of miR-203, -887, and -3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are, therefore, “tumor-suppressor-like” miRs. The combination miR-887+miR-3619 abolished >90% of PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 cells have low miR expression, high levels of PLD1/2, and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We determined that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas vimentin dampens expression of the miRs in post-EMT invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.     

PsHint1, associated with the G‐protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae

Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G‐protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1‐interacting proteins, including PsHint1, a histidine triad (HIT) domain‐containing protein orthologous to human HIT nucleotide‐binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)‐ and guanosine diphosphate (GDP)‐bound forms of PsGPA1. An analysis of the gene‐silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1‐silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα‐independent pathway involved in the pathogenicity of P. sojae.     

Platelet-Derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells

Summary Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response. Partial support for this project was obtained through a grant to C. J. W. from the American Heart Association (#88-034G) and from the W. Alton Jones Foundation.     

The switching dynamics of the bacterial flagellar motor

Many bacteria are propelled by flagellar motors that stochastically switch between the clockwise and counterclockwise rotation direction. Although the switching dynamics is one of their most important characteristics, the mechanisms that control it are poorly understood. We present a statistical–mechanical model of the bacterial flagellar motor. At its heart is the assumption that the rotor protein complex, which is connected to the flagellum, can exist in two conformational states and that switching between these states depends on the interactions with the stator proteins, which drive the rotor. This couples switching to rotation, making the switch sensitive to torque and speed. Another key element is that after a switch, it takes time for the load to build up, due to conformational transitions of the flagellum. This slow relaxation dynamics of the filament leads, in combination with the load dependence of the switching frequency, to a characteristic switching time, as recently observed. Hence, our model predicts that the switching dynamics is not only controlled by the chemotaxis‐signaling network, but also by mechanical feedback of the flagellum.     

Deciphering the hunting strategy of a bacterial wolfpack

Myxococcus xanthus is a common soil bacterium with an intricate multicellular lifestyle that continues to challenge the way in which we conceptualize the capabilities of prokaryotic organisms. Myxococcus xanthus is the preferred laboratory representative from the Myxobacteria , a family of organisms distinguished by their ability to form highly structured biofilms that include tentacle-like packs of surface-gliding cell groups, synchronized rippling waves of oscillating cells and massive spore-filled aggregates that protrude upwards from the substratum to form fruiting bodies. But most of the Myxobacteria are also predators that thrive on the degradation of macromolecules released through the lysis of other microbial cells. The aim of this review is to examine our understanding of the predatory life cycle of M. xanthus . We will examine the multicellular structures formed during contact with prey, and the molecular mechanisms utilized by M. xanthus to detect and destroy prey cells. We will also examine our understanding of microbial predator–prey relationships and the prospects for how bacterial predation mechanisms can be exploited to generate new antimicrobial technologies.     

Preliminary estimation of chemoattractant activity of staphylococcal serine proteinase in vitro

Human polymorphonuclear leukocytes isolated from peripheral blood of healthy donors migrated toward the staphylococcal serine proteinase.     

Invasive activity and chemotactic response to growth factors by Kaposi's sarcoma cells

Kaposi's sarcoma (KS) is a relatively low grade neoplasm, classically occurring in the skin of elderly men. A more virulent and invasive form of Kaposi's sarcoma has been described in patients with acquired immune deficiency syndrome (AIDS). The origin and identification of the tumor cells in these lesions is controversial. Here we have studied the behavior of cells derived from KS lesions in an in vitro assay which measures the ability of cells to invade through a reconstituted basement membrane. In agreement with previous work, KS cells obtained under selective culture conditions were invasive showing activity comparable to that of malignant tumor cells. Normal fibroblasts, smooth muscle cells, and endothelial cells did not demonstrate invasive behavior under the same experimental conditions. To characterize further the nature of the KS cells we tested the chemotactic response of cells from the most invasive line to a variety of growth factors and compared their response to those of fibroblasts, smooth muscle, and endothelial cells. These studies suggest that normal cells respond to a unique repertoire of chemotactic factors. The chemotactic response of the KS cells most closely resembled that of smooth muscle cells and was quite distinct from endothelial cells. These results indicate that the KS-derived cultures contain invasive cells with a smooth muscle cell-like phenotype.