dictyNews
Electronic Edition
Volume 35, number 20
Dec 30, 2010
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Abstracts
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The histone methyltransferase Dot1 is required for DNA damage repair and
proper development in Dictyostelium
Annette Müller-Taubenberger, Clemens Bönisch, Marlis Fürbringer,
Finni Wittek, Sandra B. Hake
BBRC, in press
Posttranslational histone modifications play an important role in modulating
gene expression and chromatin structure. Here we report the identification of
histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum.
We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate
that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1
are reduced in growth and delayed in development, but do not show apparent
changes in cell cycle regulation. Furthermore, our results indicate that Dot1
contributes to UV damage resistance and DNA repair in D. discoideum. In
summary, the data support the view that the machinery controlling the setting
of histone marks is evolutionary highly conserved and provide evidence that
D. discoideum is a suitable model system to analyze these modifications and
their functions during development and differentiation.
Submitted by Annette Müller-Taubenberger [[log in to unmask]]
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EGF-like peptide-enhanced cell motility in Dictyostelium functions independently
of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling
Robert Huber (1) and Danton H. O’Day (1,2)
(1) Department of Cell & Systems Biology, 25 Harbord Street, University of
Toronto, Toronto, ON, Canada M5S 3G5
(2) Department of Biology, University of Toronto at Mississauga,
3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6
Cellular Signalling, in press
Current knowledge suggests that cell movement in the eukaryotic slime mold
Dictyostelium discoideum is mediated by different signaling pathways involving
a number of redundant components. Our previous research has identified a
specific motility-enhancing function for Epidermal Growth Factor-Like (EGFL)
repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular,
calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP
signaling (carA-, carC-, gpaB-, gpbA-), the endogenous calcium (Ca2+) release
inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show
that DdEGFL1, a synthetic peptide whose sequence is obtained from the first
EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling
pathways to enhance cell motility through a mechanism involving Ca2+ signaling,
CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric
myosin II heavy chain and actin in the cytoskeleton by 24.1 ± 10.7% and
25.9 ± 2.1% respectively and demonstrate a link between Ca2+/CaM signaling
and cytoskeletal dynamics. Finally, our findings suggest that carA and carC
mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the
movement of carA-/carC- cells by 844 ± 136% compared to only 106 ± 6% for
parental DH1 cells. Based on our data, this signaling pathway also appears to
involve the G-protein beta subunit, RasC, RasGEFA, and protein kinase B.
Together, our research provides insight into the functionality of EGFL repeats in
Dictyostelium and the signaling pathways regulating cell movement in this
model organism. It also identifies several mechanistic components of
DdEGFL1-enhanced cell movement, which may ultimately provide a model
system for understanding EGFL repeat function in higher organisms.
Submitted by: Danton H. O’Day [[log in to unmask]]
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Tyrosine Phosphorylation of Actin during Microcyst Formation and Germination
in Polysphondylium pallidum
Aldona Budniak (1) and Danton H. O’Day (1,2)
(1) Department of Cell & Systems Biology, 25 Harbord Street, University of
Toronto, Toronto, ON, Canada M5S 3G5
(2) Department of Biology, University of Toronto at Mississauga,
3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6
Protist, in press
High osmolarity causes amoebae of the cellular slime mould Polysphondylium
pallidum to individually encyst, forming microcysts. During microcyst differentiation,
actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent
of encystment conditions and occurs during the final stages of microcyst formation.
During microcyst germination, actin undergoes dephosphorylation prior to amoebal
emergence. Renewed phosphorylation of actin in germinating microcysts can be
triggered by increasing the osmolarity of the medium which inhibits emergence.
Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in
dormant microcysts. Following the onset of germination, actin is observed around
vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin
localizes to patches near the cell surface. Increasing osmolarity disrupts this l
ocalization and causes actin to redistribute throughout the cytoplasm, a situation
similar to that observed in dormant microcysts. The tyrosine phosphorylation state
of actin does not appear to influence the long-term viability of dormant microcysts.
Together, these results indicate an association between actin tyrosine
phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.
Submitted by: Danton H. O’Day [[log in to unmask]]
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Expression, identification and purification of Dictyostelium acetoacetyl-CoA
thiolase expressed in Escherichia coli ”
Tanaka T, Shima Y, Ogawa N, Nagayama K†, Yoshida T, Ohmachi T*
Department of Biochemistry and Molecular Biology, Faculty of Agriculture and
Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, 036-8561, Japan
† Present address: Faculty of Life Sciences, University of Manchester,
Manchester, M13 9PT, UK
Int. J. Biol. Sci., in press
Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent
thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the
reverse condensation reaction. A full-length cDNA clone pBSGT3, which has
homology to known thiolases, was isolated from Dictyostelium cDNA library.
Expression of the protein encoded in pBSGT3 in Escherichia coli, its thiolase
enzyme activity, and the amino acid sequence homology search revealed that
pBSGT3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an
active form in an E. coli expression system, and purified to homogeneity by
selective ammonium sulfate fractionation and two steps of column
chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg
protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-
Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the
amino acid sequence deduced from pBSGT3 clone. The r-thiolase in the inclusion
body expressed highly in E. coli was the precursor form, which is slightly larger
than the purified r-thiolase. When incubated with cell-free extract of Dictyostelium
cells, the precursor was converted to the same size to the purified r-thiolase,
suggesting that the presequence at the N-terminus is removed by a
Dictyostelium processing peptidase.
Submitted by: Tetsuo Ohmachi [[log in to unmask]]
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Artificial compounds differentially control Dictyostelium chemotaxis and
cell differentiation.
Hidekazu Kuwayama 1, Haruhisa Kikuchi 2, Yoshiteru Oshima 2,
Yuzuru Kubohara 3
1 Graduate School of Life and Environmental Sciences, University of Tsukuba,
Tsukuba 305-8572, Japan
2 Graduate School of Pharmaceutical Sciences, Tohoku University,
Sendai 980-8578, Japan
3 Department of Molecular and Cellular Biology, Institute for Molecular and
Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan
Correspondence: Yuzuru Kubohara, Department of Molecular and Cellular
Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma
University, Maebashi 371-8512, Japan.
Cell Structure and Function, in press
Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) are small lipophilic
signal molecules that control both cell differentiation and chemotaxis in the
cellular slime mold Dictyostelium discoideum. In this study, we examined the
effects of four amide derivatives of DIF-1 on stalk cell differentiation and
chemotaxis. The DIF derivatives differentially affected cell differentiation and
chemotaxis, suggesting the possible existence of at least three receptors for
DIFs: one receptor responsible for stalk cell induction, and two receptors
responsible for chemotaxis modulation. Furthermore, our results indicate
that DIF derivatives can be utilized to analyze the DIF-signaling pathways.
Submitted by: Yuzuru Kubohara [[log in to unmask]]
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[End dictyNews, volume 35, number 20]
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