|
|
|
|
|
|
|
||
Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75235
In the biflagellated alga Chlamydomonas,
adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this ~3-µmlong fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes
during flagellar adhesion with mating type minus
(mt
) gametes. Subsequent adhesion between the tip
of the mt+ fertilization tubule and the apex of a mating
structure on mt gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this
report, we describe the isolation and characterization of
fertilization tubules from mt+ gametes activated for
cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTAcontaining buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll
gradients. As determined by fluorescence microscopy
of samples stained with a fluorescent probe for filamentous actin, the method yielded 2-3 × 106 fertilization
tubules/µg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative
stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated
mt+ gametes, retaining both the extracellular fringe
and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins
identified by biotinylation studies, copurified with the
fertilization tubules. Most importantly, the isolated
mt+ fertilization tubules bound to the apical ends of
activated mt gametes between the two flagella, the
site of the mt mating structure; a single fertilization
tubule bound per cell, binding was specific for gametes,
and fertilization tubules isolated from trypsin-treated,
activated mt+ gametes did not bind to activated mt
gametes.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
