dictyNews Electronic Edition Volume 29, number 13 November 9, 2007 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. ========= Abstracts ========= Talin influences the dynamics of the myosin VII-membrane interaction. Galdeen SA, Stephens S, Thomas DD, Titus MA Department of Genetics, Cell Biology, and Development and Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. Molecular Biology of the Cell 2007 Oct 18(10):4074-84. Myosin VII (M7) and talin are ancient and ubiquitous actin-binding proteins with conserved roles in adhesion. Talin serves to link membrane receptors to the underlying actin cytoskeleton and forms a complex with M7 in Dictyostelium. The levels of talinA are tightly linked to M7 levels in Dictyostelium. Cells lacking M7 exhibit an 80% decrease in steady-state levels of talinA, whereas increased levels of M7 result in concomitant increases in total talinA. In contrast, changes in talinA levels do not affect M7 levels. Immunoprecipitation reveals that talinA and M7 are associated with each other in membrane fractions. Fluorescence recovery after photobleaching experiments on green fluorescent protein (GFP)-M7 cells expressing different levels of the M7 and talinA show that changes in the overall amounts of these two proteins influences the dynamics of membrane-associated M7. The recovery of GFP-M7 on the membrane is faster in cells lacking talinA and limited in the presence of excess amounts of talinA and M7. These results establish that M7 stabilizes talinA in the cytosol and, in return, talinA regulates the residence time of M7 at the plasma membrane, suggesting that these two proteins are both part of the same dynamic adhesion complex on the plasma membrane. Submitted by: Shawn Galdeen [shawn.galdeen@umassmed.edu] -------------------------------------------------------------------------------- Stochastic noise and synchronisation during Dictyostelium aggregation make cAMP oscillations robust. Kim J, Heslop-Harrison P, Postlethwaite I, Bates DG PLoS Computational Biology 2007 3(11): e218. http://dx.doi.org/10.1371/journal.pcbi.0030218.eor (when this link stops working, remove 'eor' for early online release) Stable and robust oscillations in the concentration of adenosine 3', 5'-cyclic monophosphate (cAMP) are observed during the aggregation phase of starvation-induced development in Dictyostelium discoideum. In this paper we use mathematical modelling together with ideas from robust control theory to identify two factors which appear to make crucial contributions to ensuring the robustness of these oscillations. Firstly, we show that stochastic fluctuations in the molecular interactions play an important role in preserving stable oscillations in the face of variations in the kinetics of the intracellular network. Secondly, we show that synchronisation of the aggregating cells through the diffusion of extracellular cAMP is a key factor in ensuring robustness of the oscillatory waves of cAMP observed in Dictyostelium cell cultures to cell-to-cell variations. A striking and quite general implication of our results is that the robustness analysis of models of oscillating biomolecular networks (circadian clocks, Ca2+ oscillations, etc) can only be done reliably by using stochastic simulations, even in the case where molecular concentrations are very high. Submitted by: Pat Heslop-Harrison [phh4@le.ac.uk] -------------------------------------------------------------------------------- Filamin repeat segments required for photosensory signalling in Dictyostelium discoideum Sarah J Annesley, Esther Bandala-Sanchez, Afsar U Ahmed, Paul R Fisher Department of Microbiology, La Trobe University, Vic., Australia BMC Cell Biology, in press Background Filamin is an actin binding protein which is ubiquitous in eukaryotes and its basic structure is well conserved - an N-terminal actin binding domain followed by a series of repeated segments which vary in number in different organisms. D. discoideum is a well established model organism for the study of signalling pathways and the actin cytoskeleton and as such makes an excellent organism in which to study filamin. Ddfilamin plays a putative role as a scaffolding protein in a photosensory signalling pathway and this role is thought to be mediated by the unusual repeat segments in the rod domain. Results To study the role of filamin in phototaxis, a filamin null mutant, HG1264, was transformed with constructs each of which expressed wild type filamin or a mutant filamin with a deletion of one of the repeat segments. Transformants expressing the full length filamin to wild type levels completely rescued the phototaxis defect in HG1264, however if filamin was expressed at lower than wild type levels the phototaxis defect was not restored. The transformants lacking any one of the repeat segments 2-6 retained defective phototaxis and thermotaxis phenotypes, whereas transformants expressing filaminDelta1 exhibited a range of partial complementation of the phototaxis phenotype which was related to expression levels. Immunofluorescence microscopy showed that filamin lacking any of the repeat segments still localised to the same actin rich areas as wild type filamin. Ddfilamin interacts with RasD and IP experiments demonstrated that this interaction did not rely upon any single repeat segment or the actin binding domain. Conclusions This paper demonstrates that wild type levels of filamin expression are essential for the formation of functional photosensory signalling complexes and that each of the repeat segments 2-6 are essential for filamins role in phototaxis. By contrast, repeat segment 1 is not essential provided the mutated filamin lacking repeat segment 1 is expressed at a high enough level. The defects in photo/thermosensory signal transduction caused by the absence of the repeats are due neither to mislocalisation of filamin nor to the loss of RasD recruitment to the previously described photosensory signalling complex. Submitted by: Paul Fisher [P.Fisher@latrobe.edu.au] -------------------------------------------------------------------------------- Dictyostelium Extracellular Vesicles Containing Hoechst 33342 Transfer the Dye into the Nuclei of Living Cells: A Fluorescence Study Irene Tatischeff, Francoise Lavialle, Sophie Pigaglio-Deshayes, Christine Pechoux-Longin, Laurent Chinsky, Annette Alfsen Journal of Fluorescence, in press Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476–87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed. Submitted by: Irene Tatischeff [tati@ccr.jussieu.fr] ============================================================== [End dictyNews, volume 29, number 13]