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

(TPM-1), a subunit of the Arp2/3 complex (ARP-3) and coronin (COR1).FIM-GFP, ARP-3-GFP, and COR1-GFP associated with small patches inthe cortical cytoplasm that were concentrated in a subapical ring. Thesecortical patches were short-lived, and a subset was mobile throughout thehypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP andLifeact-GFP co-localized within the Spitzenkörper core at the hyphal apex,and were also observed in actin cables throughout the hypha. All GFP fusionproteins studied were also transiently localized at septa: Lifeact-GFP firstappeared as a broad ring during early stages of contractile ring formationand later coalesced into a sharper ring, TPM-1-GFP was observed inmaturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed adouble ring flanking the septa. Our observations suggest that each of the N.crassa F-actin-binding proteins analyzed associates with a different subset ofF-actin structures, presumably reflecting distinct roles in F-actinorganization and dynamics during all the stages of development andseptation. Actin is present since early stages of septum formation, thecontractile force of the actomyosin ring is related to the presence oftropomyosin and it seems that there is a need of plasma membraneremodeling regards the presence of endocytic patches labeled by fimbrin,coronin and Arp2/3 complex.CBV004Interaction of bacterial cytoskeletal elements in aheterologous systemF. Dempwolff*, C. Reimold, P.L. GraumannDepartment of Microbiology, Albert-Ludwigs-University, Freiburg,GermanyBacterial cytoskeletal element MreB has been shown to be essential for themaintenance of rod cell shape in many bacteria. MreB forms rapidlyremodelling helical filaments underneath the cell membrane in Bacillussubtilis and other bacterial cells, and co-localizes with its two paralogs, Mbland MreBH. We show that MreB localizes as dynamic bundles of filamentsunderneath the cell membrane in Drosophila S2 Schneider cells, whichbecome highly stable when the ATPase motif was modified. Extendedinduction of MreB resulted in the formation of membrane protrusions,showing that like actin, MreB can exert force against the cell membrane.Mbl also formed membrane associated filaments, while MreBH formedfilaments within the cytosol. When co-expressed, MreB, Mbl and MreBHbuilt up mixed filaments underneath the cell membrane. RodZ membraneprotein localized to internal membranes in S2 cells, but localized to the thecell membrane when co-expressed with Mbl, showing that membraneassociated structures can recruit a membrane protein. Thus, MreB paralogsform a self-organizing filamentous scaffold underneath the membrane that isable to recruit other proteins to the cell surface.CBV005The Kinesin-3 Motor Protein UncA Reveals DifferentMicrotubule Populations in Aspergillus nidulansN. Zekert*, C. Seidel, R. FischerDepartment of Microbiology, Karlsruhe Institute of Technology, Karlsruhe,GermanyThe MT cytoskeleton is not as rigid and uniform as the name implies, but ischaracterized by its dynamic instability. In addition, MTs can be made up ofdifferent tubulin isoforms and can be post-translationally modified. MTmodifications are evolutionarily old „inventions” and occur in primitiveeukaryotes such as Giardia lamblia, whereas detyrosination appeared laterduring evolution. Here we found that the A. nidulans kinesin-3, UncA,transports vesicles along microtubules (MTs) and is required for hyphalextension. Most surprisingly, UncA-dependent vesicle movement occurredalong a subpopulation of MTs. GFP labelled UncA rigor decorated a singleMT bundle, which remained intact during mitosis, while other cytoplasmicMTs were depolymerised. Mitotic spindles were not labelled with GFP-UncA rigor but reacted with a specific antibody against tyrosinated alphatubulin.Those results suggest that UncA binds preferentially todetyrosinated MTs [1] and that different MT populations exist in A.nidulans. To confirm this aim we searched for the MT modification enzyme- tubulin tyrosine ligase (TtlA) - and constructed a ttlA-deletion strain and attlA, alpha tubulin 2 (tubB) double deletion strain. Currently we arecharacterizing the MT cytoskeleton and its modification in the wild typestrain and in the deletion strains using different assays and techniques.To understand how UncA is able to distinguish between different MTs,deletion analyses revealed a specificity region in the tail of UncA betweenamino acid 1316 and 1402. A non-targeted Y2H approach was used toidentify interaction partners of this region, which are most likely involved inrecognition of MT subpopulations. Two candidates appeared to beassociated with vesicles and currently different assay are performed toconfirm their interaction with UncA.[1] Zekert, N. and R. Fischer (2009): Mol. Biol. Cell 20, 673-684.CBV006Physical compartmentalization by a protein diffusionbarrier in stalked alpha-proteobacteriaS. Schlimpert* 1,2 , A. Briegel 3 , K. Bolte 2 , U.G. Maier 2 , J. Kahnt 4 ,G.J. Jensen 3 , M. Thanbichler 1,21 Research Group Prokaryotic Cell Biology, Max Planck Insitute forTerrestrial Microbiology, Marburg, Germany2 Department of Biology, Philipps-University, Marburg, Germany3 Division of Biology and Howard Hughes Medical Institute, CaliforniaInstitute of Technology, Pasadena, USA4 Department of Ecophysiology, Max Planck Institute for TerrestrialMicrobiology, Marburg, GermanyProsthecae, also known as stalks, are a widespread phenomenon amongbacteria, but the biogenesis and function of these structures is still unclear.In the dimorphic alpha-proteobacterium Caulobacter crescentus, the stalkrepresents a thin extension of the cell envelope that is free of DNA,ribosomes and most cytoplasmic proteins. It is segmented at irregularintervals by so-called crossbands, disk-like structures that traverse the entirewidth of the stalk perpendicular to the long-axis of the cell. Crossbands havebeen observed in a variety of prosthecate species and are generally thoughtto have an architectural, stabilizing function.Here, we report the identification and characterization of four novel stalkproteins, StpABCD, that are essential for crossband formation and stalkcompartmentalization in Caulobacter. Synthesis of StpABCD is initiated atthe onset of stalk outgrowth, an obligate and irreversible developmentalcheckpoint in the Caulobacter cell-cycle. We found that StpABCD arespecifically targeted to the periplasmic space of the stalk, with StpA actingas a recruitment factor for StpBCD. The four proteins colocalize in distinctfoci that display the same subcellular distribution as crossbands.Additionally, coimmunoprecipitation analysis supports the idea thatStpABCD interact in vivo to form a multi-protein complex. Electron cryotomographyrevealed that cells deficient in StpAB consistently lackcrossbands. We used fluorescence-recovery after photobleaching (FRAP) totest for the presence or absence of protein compartmentalization in wild-typeand StpAB-deficient cells. Interestingly, our experiments demonstrated thatcrossbands act as diffusion barriers for periplasmic and inner membraneproteins. However, the mechanism by which cytosolic proteins are retainedin the cell-body is still unclear. We are currently examining whether thefunction of crossbands is conserved among stalked alpha-proteobacteria.Based on our findings, we hypothesize that StpABCD are required forsynthesizing crossbands, which act as a protein diffusion barrier tocompartmentalize the periplasmic space of the stalk and physically separateit from the cell-body. Crossband formation thus represents a novelmechanism to topologically restrict protein mobility within a cell.CBV007Microtubule-dependent co-transport of mRNPs andvesicles in Ustilago maydisT. Pohlmann* 1,2 , S. Baumann 1,2 , M. Jungbluth 2,3 , M. Feldbrügge 11 Institute for Microbiology, Heinrich-Heine-University, Duesseldorf,Germany2 Department for Organismic Interactions, Max Planck Institute forTerrestrial Microbiology, Marburg, Germany3 Institute for Molecular Genetics, Philipps-University, Marburg, GermanyLong-distance transport of mRNAs is important in determining polarity ineukaryotes. In U. maydis this process is mediated by the RNA bindingprotein Rrm4 which is a key component of large motile ribonucleoproteincomplexes (mRNPs) shuttling along the microtubule cytoskeleton.Disruption of long-distant mRNP transport by deleting or mutating rrm4leads to defects in filamentous growth and a reduced virulence. In spite ofidentifying numerous transported mRNAs which encode upon otherspolarity and translation factors, the composition of the mRNPs and themotor proteins involved in their transport were not known. Here we showthat the plus end-directed conventional kinesin Kin1, the UNC104/Kif1A-spektrum | Tagungsband 2011