dictyNews
Electronic Edition
Volume 41, number 1
January 9, 2015

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Abstracts
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Evidence for nucleolar subcompartments in Dictyostelium

Andrew Catalano (a) and Danton H. O’Day (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. O’Day: [log in to unmask]
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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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 [[log in to unmask]]   
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[End dictyNews, volume 41, number 1]