dictyNews
Electronic Edition
Volume 32, number 3
January 30, 2009

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Abstracts
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The 3-Dimensional Dynamics of Actin Waves,
a Model of Cytoskeletal Self-organization

Till Bretschneider1, 3,§, Kurt Anderson2, 5,§, Mary Ecke1,
Annette Müller-Taubenberger1, 4,  Britta Schroth-Diez2,
Hellen C. Ishikawa-Ankerhold1, and Günther Gerisch1*

1 Max-Planck-Institut für Biochemie, Am Klopferspitz 18,
D-82152 Martinsried, Germany.
2 Max-Planck-Institut für molekulare Zellbiologie und Genetik,
Pfotenhauerstrasse 108, D 01307 Dresden, Germany.
3 Present address: Warwick Systems Biology, Coventry House,
University of Warwick, Coventry CV4 7AL, UK.
4 Present address: Adolf Butenandt Institute / Cell Biology and Munich
Center for Integrated Protein Science (CIPSM), Ludwig Maximilians  
Universität,
Schillerstrasse 42, D-80336 München, Germany.
5 Present address: Leader Light Microscopy, Beatson Institute for Cancer
Research, Garscube Estate, Switchback Road, Glasgow, G61 1BD, Scotland  
UK.
§ These two authors contributed equally to the work.

*Correspondence should be addressed to Dr. Günther Gerisch,
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
phone: (+49) 89-8578-2326; fax: (+49) 89-8578-3885;
e-mail: [log in to unmask]


Biophys.J., in press.

Actin polymerization is typically initiated at specific sites in a  
cell by
membrane-bound protein complexes, and the resulting structures
are involved in specialized cellular functions, such as migration,
particle uptake, or mitotic division. Here we analyze the potential
of the actin system to self-organize into waves that propagate on
the planar, substrate-attached membrane of a cell. We show that
self-assembly involves the ordered recruitment of proteins from
the cytoplasmic pool, and relate the organization of actin waves
to their capacity for applying force. Three proteins are shown to
form distinct 3-dimensional patterns in the actin waves. Myosin-IB
is enriched at the wave front and close to the plasma membrane,
the Arp2/3 complex is distributed throughout the waves, and
coronin forms a sloping layer on top of them. CARMIL, a protein
that links myosin-IB to the Arp2/3 complex, is also recruited to the
waves. Wave formation does not depend on signals transmitted
by heterotrimeric G-proteins, nor does their propagation require
SCAR, a regulator upstream of the Arp2/3 complex. Propagation
of the waves is based on an actin treadmilling mechanism,
indicating a program that couples actin assembly to disassembly
in a 3-dimensional pattern. When waves impinge on the cell
perimeter, they push the edge forward, when they reverse direction,
the cell border is paralyzed. These data show that force-generating,
highly organized supramolecular networks are autonomously
formed in live cells from molecular motors and proteins controlling
actin polymerization and depolymerization.


Submitted by: Günther Gerisch [[log in to unmask]]
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A measure of endosomal pH by flow cytometry in Dictyostelium

Anna Marchetti, Emmanuelle Lelong and Pierre Cosson

BMC Research Notes 2009, 2:7

Dictyostelium amoebae are frequently used to study the organization
and function of the endocytic pathway, and specific protocols are
essential to measure the dynamics of endocytic compartments and
their internal pH. We have revisited these classical protocols to  
measure
more accurately endosomal pH, making use of a fluorescent probe
(Oregon green) more adequate for very acidic pH values. This
pH-sensitive probe was combined with a pH-insensitive marker,
in order to visualize simultaneously endosome dynamics and pH
changes. Finally, a flow cytometer was used to measure endosomal
pH in individual cells. Using these simple protocols the endosomal
pH of endocytic compartments can be assessed accurately, revealing
the extreme acidity of Dictyostelium lysosomes (pH <3.5).


Submitted by: Emmanuelle Lelong [[log in to unmask]]
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[End dictyNews, volume 32, number 3]