dictyNews Electronic Edition Volume 38, number 25 September 28, 2012 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains Khalid Arhzaouy1, Karl-Heinz Strucksberg1,2, Sze Man Tung1, Karthikeyan Tangavelou1, Maria Stumpf1, Jan Faix3, Rolf Schroeder2, Christoph S. Clemen1, and Ludwig Eichinger1 1Intitute for Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany 2Institute of Neuropathology, University Hospital Erlangen, Erlangen, Germany 3Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany PLos ONE, in press Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97R155C) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9KO) cells. Native gel electrophoresis showed that both p97 and p97R155C assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9KO/p97R155C-RFP and ATG9KO cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9KO mutant. A major finding is that the expression of p97R155C-RFP in the ATG9KO strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition. Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de] --------------------------------------------------------------------------- Fast Characterization of Cell-Derived Extracellular Vesicles by Nanoparticles Tracking Analysis, Cryo-Electron Microscopy and Raman Tweezers Microspectroscopy Irene Tatischeff1*, Eric Larquet2, Juan M. Falcon-Perez3, Pierre-Yves Turpin1 and Sergei G. Kruglik1 Journal of Extracellular Vesicles, in press The joint use of three complementary techniques, i.e. Nanoparticles Tracking Analysis, Cryo-Electron Microscopy and Raman Tweezers Microspectroscopy, is proposed for a rapid characterization of extracellular vesicles of various origins. Nanoparticles Tracking Analysis is valuable for studying the size distribution and concentration, Cryo-Electron Microscopy is outstanding for the morphological characterization, including observation of vesicles heterogeneity, while Raman Tweezers Microspectroscopy provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic extracellular vesicles. At least two separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the two physiological states of this non-pathogenic microorganism, i.e. cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of Raman Tweezers Microspectroscopy. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterization of extracellular vesicles for the purpose of human diseases diagnosis. Submitted by Irene Tatischeff [irene.tatischeff@upmc.fr] --------------------------------------------------------------------------- Rules of engagement: centrosome-nuclear connections in a closed mitotic system Meredith Leo, Diana Santino, Irina Tikhonenko, Valentin Magidson, Alexey Khodjakov, and Michael P. Koonce. Biology Open, in press The assembly of a functional mitotic spindle is essential for cell reproduction and requires a precise coordination between the nuclear cycle and the centrosome. This coordination is particularly prominent in organisms that undergo closed mitosis where centrosomes must not only respond to temporal signals, but also to spatial considerations, e.g. switching from the production of cytoplasmic microtubule arrays to the generation of dynamic intra-nuclear microtubules required for spindle assembly. We utilize a geneknockout of Kif9, a Dictyostelium discoideum Kin-I kinesin, to destabilize the physical association between centrosomes and the nuclear envelope. This approach presents a unique opportunity to reveal temporal and spatial components in the regulation of centrosomal activities in a closed-mitosis organism. Here we report that centrosome-nuclear engagement is not required for the entry into mitosis. Although detached centrosomes can duplicate in the cytoplasm, neither they nor nuclei alone can produce spindle-like microtubule arrays. However, the physical association of centrosomes and the nuclear envelope is required to progressthrough mitosis beyond prometaphase. Submitted by Michael Koonce [koonce@wadsworth.org] ============================================================== [End dictyNews, volume 38, number 25]