Regulation of polarised growth in fungi

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkörper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkörper. The Spitzenkörper then acts as vesicle supply centre in which vesicles exit the Spitzenkörper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkörper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research

Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKC alpha, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKC alpha directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residueopen

Antineoplastic Drugs as a Potential Risk Factor in Occupational Settings: Mechanisms of Action at the Cell Level, Genotoxic Effects, and Their Detection Using Different Biomarkers

U članku je prikazana osnovna podjela antineoplastičnih lijekova prema mehanizmima djelovanja na razini stanice. Objašnjeni su mehanizmi genotoksičnosti najvažnijih vrsta lijekova koji se primjenjuju u okviru uobičajenih protokola za liječenje zloćudnih novotvorina. Navedena je važeća klasifi kacija antineoplastika prema kancerogenom potencijalu, podaci o mutagenom potencijalu te je prikazana njihova podjela u skladu s anatomsko-terapijsko-kemijskim sustavom klasifi kacije. Sustavno su prikazani najvažniji rezultati svjetskih i hrvatskih istraživanja na populacijama radnika izloženih antineoplasticima, provedenih u razdoblju 1980.-2009. s pomoću četiri najčešće primjenjivane metode: analize izmjena sestrinskih kromatida, analize kromosomskih aberacija, mikronukleus-testa i komet-testa. Objašnjena su osnovna načela navedenih metoda te raspravljene njihove prednosti i nedostaci. Biološki pokazatelji daju važne podatke o individualnoj osjetljivosti profesionalno izloženih ispitanika koji mogu poslužiti unaprjeđenju postojećih uvjeta rada i upravljanju rizicima pri izloženosti genotoksičnim agensima. Na osnovi prednosti i nedostataka citogenetičkih metoda zaključeno je da je mikronukleus-test, koji podjednako uspješno dokazuje klastogene i aneugene učinke, jedna od najboljih metoda dostupnih za otkrivanje štetnih djelovanja antineoplastičnih lijekova koji su u aktivnoj primjeni.This article brings an overview of the mechanisms of action of antineoplastic drugs used in the clinical setting. It also describes the genotoxic potentials of the most important classes of antineoplastic drugs involved in standard chemotherapy protocols. Classifi cation of antineoplastic drugs according to the IARC monographs on the evaluation of carcinogenic risks to humans is accompanied by data on their mutagenicity and the most recent updates in the Anatomical Therapeutic Chemical (ATC) Classifi cation System. We report the main fi ndings of biomonitoring studies that were conducted in exposed healthcare workers all over the world between 1980 and 2009 using four biomarkers: sister chromatid exchanges, chromosome aberrations, micronuclei. and the comet assay. The methods are briefl y explained and their advantages and disadvantages discussed. Biomarkers provide important information on individual genome sensitivity, which eventually might help to improve current working practices and to manage the risks related with exposure to genotoxic agents. Taking into consideration all known advantages and drawbacks of the existing cytogenetic methods, the micronucleus assay, which is able to detect both clastogenic and aneugenic action, is the most suitable biomarker for assessing harmful effects of antineoplastic drugs currently used in health care

The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst

Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway