|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1997//1537 $5.00
The Journal of Cell Biology, Volume 137, Number 7,
, 1997 1537-1553
Article |
The Chlamydomonas Mating Type Plus Fertilization Tubule, a Prototypic Cell Fusion Organelle: Isolation, Characterization, and In Vitro Adhesion to Mating Type Minus Gametes
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-µm-long 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 EGTA-containing 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 x 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.
Abbreviations used in this paper: ft, fertilization tubule; FTSB, fertilization tubule stabilization buffer; mt, mating type; N-free medium, nitrogen-free medium.
Address all correspondence to William J. Snell, Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX 75235. Tel.: (214) 648-2332. Fax: (214) 648-8694.
This work was submitted in partial fulfillment of the requirements for the Ph.D. degree for Nedra F. Wilson, University of Texas Southwestern Graduate School of Biomedical Sciences, Dallas, TX.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
