dictyNews Electronic Edition Volume 41, number 1 January 9, 2015 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 ========= Evidence for nucleolar subcompartments in Dictyostelium Andrew Catalano (a) and Danton H. OĠDay (a,b)* a Department of Biology, University of Toronto at Mississauga, 3359 Mississauga rd. N., Mississauga, Ontario, Canada, L5L 1C6 b Department of Cell and Systems Biology, University of Toronto, 25 Harbord st., Toronto, Ontario, Canada, M5S 3G5 Biochemical Biophysical Research Communications, in press Free online: http://authors.elsevier.com/sd/article/S0006291X14022165 The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during nucleolar disruption as a result of either AM-D treatment or mitosis support these subcompartments. A model for the AM-D-induced redistribution patterns is proposed. Submitted by: Danton H. OĠDay: danton.oday@utoronto.ca ---------------------------------------------------------------------- Partial genetic suppression of a loss of function mutant of the Neuronal Ceroid Lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum Jonathan E. Phillips and Richard H. Gomer Disease Models & Mechanisms, in press Neuronal Ceroid Lipofuscinosis (NCL) is the most common childhood- onset neurodegenerative disease. NCL is inevitably fatal, and there is no current treatment. Children with NCL show progressive decline in movement, vision, and mental abilities and accumulation of autofluorescent deposits in neurons and other cell types. Late- infantile NCL is caused by mutations in the lysosomal protease tripeptdyl peptidase 1 (TPP1). TPP1 cleaves tripeptides from the N-terminus of proteins in vitro, but little is known about the physiological function of TPP1. TPP1 shows wide conservation in vertebrates but is not found in Drosophila, C. elegans, or S. cerevisiae. Here, we characterize ddTpp1, a TPP1 ortholog present in the social amoeba Dictyostelium discoideum. Lysates from cells lacking ddTpp1 show reduced but not abolished ability to cleave a TPP1 substrate, suggesting that other Dictyostelium enzymes can perform this cleavage. ddTpp1 and human TPP1 localize to the lysosome in Dictyostelium, indicating conserved function and trafficking. Cells lacking ddTpp1 show precocious multicellular development and a reduced ability to form spores during development. When cultured in autophagy-stimulating conditions, cells lacking ddTpp1 rapidly decrease in size and are less viable than wild-type cells, suggesting that one function of ddTpp1 may be to limit autophagy. Cells lacking ddTpp1 show strongly impaired development in the presence of the lysosome-perturbing drug chloroquine, and this phenotype can be suppressed by a secondary mutation in the gene stpA, which encodes a protein with some similarity to mammalian oxysterol-binding proteins (OSBPs). Together, these results suggest that targeting specific proteins may be a viable way to suppress the effects of loss of TPP1 function. Submitted by Richard Gomer [rgomer@tamu.edu] ---------------------------------------------------------------------- Compact Halo-Ligand-Conjugated Quantum Dots for Multicolored Single-Molecule Imaging of Overcrowding GPCR Proteins on Cell Membranes. Akihito Komatsuzaki, Tatsuya Ohyanagi, Yoshikazu Tsukasaki, Yukihiro Miyanaga, Masahiro Ueda, and Takashi Jin Small. 2014 Dec 12. doi: 10.1002/smll.201402508. http://onlinelibrary.wiley.com/doi/10.1002/smll.201402508/abstract To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved. Submitted by Yukihiro Miyanaga [miyang@bio.sci.osaka-u.ac.jp] ---------------------------------------------------------------------- Surcel A, Ng W-P, West-Foyle H, Zhu Q, Ren Y, Avery L, Krenc AK, Meyers D, Rock RS, Anders RA, Freel Meyers C, Robinson DN. Pharmacological activation of myosin II paralogs to correct cell mechanics defects. Proc. Natl. Acad. Sci. USA 2015, in press Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human MYH10 and MYH14, but not MYH9. We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof-of-concept that cell mechanics offers a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. Submitted by Doug Robinson [dnr@jhmi.edu] ============================================================== [End dictyNews, volume 41, number 1]