VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

in all directions, generating a sharp gradient of exocytosis, with a maximumat the pole and vanishing gradually in the subapex. Those secretory vesiclesreaching the plasma membrane, prior to SNAREs recognition, arepresumably tethered to their target acceptor membrane in a process mediatedby the exocyst complex. We endogenously tagged with GFP the exocystcomponents SEC-3, SEC-5, SEC-6, SEC-8, SEC-15, EXO-70 and EXO-84in N. crassa. Some components accumulated surrounding the frontal part ofthe Spitzenkörper, while others were found in a delimited region of theapical plasma membrane that correlates with the place of intensiveexocytosis during polarized growth. A more detailed analysis by TIRFMrevealed that the fluorescently labeled exocyst components followed apulsatile exocytotic process, suggesting anorderly mechanism for exocytosisof the vesicles constituting the Spitzenkörper. Our results show that theregion of exocyst-mediated vesicle fusion at the hyphal apical plasmamembrane has the same extension than the exocytosis gradient predictedearlier by the VSC model.CBV012Physical organization and interactions between sensoryhistidine kinases in E. coliE. Sommer* 1 , A. Vaknin 2 , A. Müller 1 , V. Sourjik 11 Center for Molecular Biology (ZMBH), DKFZ-ZMBH-Alliance, Universityof Heidelberg, Heidelberg, Germany2 Racah Institute of Physics, Hebrew University, Jerusalem, IsraelMicroorganisms commonly use ‘two-component’ signaling systems forsensing environmental conditions. Prototypical two-component systems arecomprised of a sensory histidine kinase and a response regulator that isphosphorylated by the kinase and typically acts as a transcription factor.Apart from a few well-investigated cases, such as signaling in bacterialchemotaxis or asymmetric cell division in bacteria, intracellular organizationof sensory kinases remains largely unclear. We characterize the spatialdistribution and oligomeric state of these sensors in the model bacteriumEscherichia coli, using fluorescence imaging, fluorescence resonance energytransfer (FRET) and fluorescence polarization microscopy. We find that atphysiological expression levels most fluorescently tagged sensors show auniform membrane distribution with no preference towards polar regions, afew kinases exhibit lateral localization patterns. Measurements of FRETconfirmed that at physiological expression levels most sensors self-associateto form small complexes, presumably dimers, but not larger oligomers. Wedemonstrate that in some of the cases interactions between sensors aresensitive to specific stimulation, suggesting that changes in proteinarrangement play a role in signal processing. We further observed severalcases of mixed complex formation between different sensors, indicatinginterconnections between different signaling pathways. However, differentfrom the signaling in chemotaxis, only few two-component sensors showeda distinct punctuate localization in the cell or low levels of fluorescencepolarization that are indicative of higher-order complexes, suggesting that atlow expression levels most of them function as isolated dimers.CBV013Understanding long-range endosome trafficking: Frommeasuring to modellingM. Schuster 1 , S. Kilaru 1 , C. Lin 2 , P. Ashwin 2 , N.J. Severs 3 , G. Steinberg* 11 Department of Biosciences, University of Exeter, Exeter, United Kingdom2 Mathematics Research Institute, University of Exeter, Exeter, UnitedKingdom3 Heart and Lung Institute, Imperial College London, London, UnitedKingdomIn filamentous fungi microtubules form long tracks that are used bymolecular motors to transport organelles, vesicles and RNA over longdistances. Such membrane trafficking is essential for hyphal tip growth, andthe underlying molecular machinery is conserved amongst filamentousfungi. The fungal pathogen Ustilago maydis is a genetically tractable systemto investigate motor cooperation in trafficking of early endosomes. Imagingof native levels of motors and their cargo in living cells in combination withquantitative analysis and mathematical modelling revealed that acombination of stochastic motor behaviour and active retention concentratedynein at microtubule ends. This is essential for efficient cargo loading andretrograde transport of early endosomes.CBV014A synthetic in vivo system identifies a chromosometethering factor in Corynebacterium glutamicum.C. Donovan*, R. Krämer, M. BramkampInstitute for Biochemistry, University of Cologne, Cologne, GermanyThe chromosome partitioning system of the rod-shaped actinomycete,Corynebacterium glutamicum consists of the Walker-type ATPase ParA, theDNA-binding protein ParB and parS sites that are found near thechromosomal origin of replication. Once chromosome replication has beeninitiated, the C. glutamicum ParB protein specifically binds the parS sites ofthe newly replicated oriC. As the chromosome is replicated, ParA binds theParB-parS nucleoprotein complex, and is thought to provide the drivingforce to relocalize the replicating chromosome to the opposite cell pole. Thechromosome is then stably attached to the cell pole, where it remains and thecell divides in between the segregated chromosomes. We were interested inidentifying and analyzing the chromosome polar targeting factor. Onepossible candidate for tethering the chromosome to the cell poles is theDivIVA protein, which influences apical growth and cell shapedetermination in C. glutamicum, similar to other organisms likeStreptomyces coelicolor and Mycobacterium smegmatis. Indeed, bacterialtwo-hybridanalysis showed an interaction between DivIVA and the Parproteins. However, to further analyse these interactions, a synthetic in vivoapproach was developed. In this system, E. coli cells are used as a host forexpression of the fluorescently labeled proteins. E. coli is advantageous forthis purpose as it does not contain homologues of the Par system or DivIVA.When expressed individually, DivIVA-GFP localized to the curved polarmembranes and division sites, while ParB-CFP showed no specificlocalization. However, upon co-expression, ParB-CFP was completelyrecruited to the polar and septal localized DivIVA. Using this system, alongwith mutational analysis the interaction sites between ParB and DivIVAcould be mapped. Also, similar interaction studies were also carried out forthe notorious pathogen Mycobacterium tuberculosis, showing that thissystem is a general mechanism within the Corynebacterianae.CBV015The Num1 Protein of Ustilago maydis is Required forPolar and Filamentous GrowthN. Kellner*, K. Heimel, J. KämperInstitute for Applied Biosciences, Genetics, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, GermanyIn the corn smut fungus Ustilago maydis, sexual development is initiated bythe fusion of two haploid sporidia, resulting in a filamentous growingdikaryon that is capable to infect the host plant. Growth of the dikaryonrequires an elaborate regulation of the cell cycle, migration and distributionof the two nuclei and the polar growth of the hyphae.We have identified the Num1 protein with a pivotal function during theseprocesses. Num1 is a homologue of SPF27, one of the core components ofthe highly conserved Prp19/CDC5 splicing associated complex. Vegetativegrowth of sporidia is not altered in num1 deletion mutants; however, thehyphae show various polarity defects, delocalized septae and dislocalizednuclei. Using the Yeast Two-Hybrid system, we identified CDC5, anotherconserved component of the Prp19/CDC5 complex, as Num1 interactor.Interestingly, we also identified various proteins with functions duringvesicle-mediated transport, in particular the kinesin 1 motor protein. TheNum1/Kin1 interaction was verified by Co-Immunoprecipitation and Split-GFP analysis. Both num1 and kin1 deletion strains exhibit identicalphenotypes with respect to vacuole morphology, filamentous and polarapical growth, corroborating the genetic interaction between Num1 andKin1.Our data connect the splicing machinery and long distant transport in U.maydis. We will present our current view whether (and how) these twodisparate mechanisms may be matched.spektrum | Tagungsband 2011