CSM News
Electronic Edition
Volume 4, number 9
March 11, 1995

Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu.

Back issues of CSM-News, the CSM Reference database and other useful
information is available by anonymous ftp from worms.cmsbio.nwu.edu
[165.124.233.50], via Gopher at the same address, or by World Wide Web
at the URL "http://worms.cmsbio.nwu.edu/dicty.html"

===============
 Announcements
===============

Dietmar Manstein has deposited the sequences of several new and
potentially useful Dicty vectors with the archive.  These sequences
are available in the directory /pub/dicty/Vector_Sequences

===========
 Abstracts
===========

OVEREXPRESSION  OF MYOSIN MOTOR DOMAINS IN DICTYOSTELIUM DISCOIDEUM: 
SCREENING OF TRANSFORMANTS AND PURIFICATION OF THE AFFINITY TAGGED 
PROTEIN

Dietmar J. Manstein and Deborah M. Hunt

National Institute for Medical Research, The Ridgeway, Mill Hill, 
London NW7 1AA, U.K.

Journal of Muscle Research and Cell Motility, in press

Summary

   The eukaryotic organism Dictyostelium discoideum has become one of
the organisms of choice for the over-expression of recombinant myosins
and myosin fragments.  Here, we describe a protocol that facilitates
the screening of cells that have been transformed with myosin
expression constructs and allows the rapid purification of recombinant
myosins.
   Depletion of cellular ATP is used to recruit most of the endogenous
and recombinant myosin into a rigor-like complex with actin. Following
cell lysis the insoluble actomyosin complex is precipitated by
centrifugation, washed, and Mg2+-ATP is added to extract the
recombinant protein from the pellet. More than 90% of the protein in
the resulting supernatant corresponds to actin, myosin, and the
recombinant myosin fragments. Therefore, it is easy to detect any
differences in expression level between individual myosin constructs
on SDS-polyacrylamide gels.  Additionally, the dependence of
expression on external factors, such as cell density, can be readily
determined.  Furthermore, the presence of a band corresponding to the
recombinant protein indicates that the overexpressed protein has at
least some of the functional properties that are characteristic for a
myosin motor.  This rapid and selective extraction protocol can also
be utilised to facilitate the purification of recombinant myosin
motors on a preparative scale and has proved particularly useful in
the purification of myosin head fragments, that are tagged with
histidine residues, by Ni2+-chelate chromatography.
 
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Amoeboid gametes and fertilization in Dictyostelium: Gamete and
pronuclear fusion are mediated by calmodulin and its binding proteins

Danton H. O'Day, Keith E. Lewis, Michael A. Lydan

Department of Zoology, University of Toronto, Erindale College, 3359
Mississauga Road, Mississauga, ON L5L 1C6 CANADA

In: Advances in Spermatozoal Taxonomy and Phylogeny. B.G.M. Jamieson,
J. Ausio & J.-L. Justine (Editors), Memoires du Museum National
d'Histoire Naturelle, Paris (1995)

Abstract

   The gametes of Dictyostelium discoideum are tiny amoeboid cells
which contain a small condensed nucleus. Gamete formation is
calcium-independent and inhibited by calmodulin function. Accumulated
data suggests that the gametes are a source of sexual pheromone that
promotes sexual cell fusion. Time-lapse videomicroscopy has revealed
that gametes are highly motile compared to non-gametic cells and, when
two gametes contact, fusion results in the formation of a binucleate
cell. Within each binucleate, pronuclear migration, swelling and
fusion occur as the cytoplasmic volume of the cell increases
dramatically producing a zygote giant cell. Gamete fusion is
calcium-dependent and involves at least one membrane-bound,
GlycNAc-containing glycoprotein (gp138). Fertilization is mediated by
the dual signal transduction pathway involving calcium and protein
kinase C. Of particular importance is the downstream role of
calmodulin (CaM) and its binding proteins (CaMBPs). A putative CaM
Kinase III activity and two, as yet unidentified CaMBPs (i.e.,
CaMBP-48, CAMBP-91) are developmentally regulated and temporally
associated with the events of cell and pronuclear fusion in D.
discoideum. Fertilization is terminated by a feed-back mechanism
involving an autoinhibitor that is secreted by the zygote giant cells.
This low molecular weight, hydrophoic, heat-stable autoinhibitor
inhibits both cell and pronuclear fusion by preventing the interaction
of CaM with its binding proteins. These results are discussed in terms
of fertilization and signal transduction involving calmodulin and its
binding proteins in higher animals.

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[End CSM News, volume 4, number 9]