Elongation of the fertilization tubule in Chlamydomonas: new observations on the core microfilaments and the effect of transient intracellular signals on their structural integrity

Experimental manipulations of gametes of Chlamydomonas reinhardi and ultrastructural observation were used to examine the composition of the microfilaments in the fertilization tubule, their probable mode of formation, and their interaction with intracellular signals. Decoration with myosin subfragment-1 was used to demonstrate that the microfilaments in the fertilization tubule were actin filaments having uniform polarity: Myosin subfragment-1 arrowheads pointed away from the membrane at the tip of the process. Filaments were attached to the cone- shaped "doublet zone" at the base of the process by their pointed ends. Discrete attachment sites for filaments on the surface of the doublet zone were seen in stereo view. To test whether actin polymerization might accompany elongation of the fertilization tubule, mating gametes were exposed to cytochalasin D in an attempt to block actin polymerization. Treatment of mating type "plus" gametes with cytochalasin D prior to and during mating inhibited the appearance of actin filaments in fertilization tubules, suppressed fertilization tubule outgrowth, and lowered mating efficiency from 90 to 15%. The role of signals generated by flagellar adhesion in maintaining the structural integrity of the microfilament-doublet zone complex was examined by correlating flagellar disadhesion with the kinetics of breakdown of the complex. In zygotes, where flagellar disadhesion occurred after cell fusion, the complex disassembled within 3 h after mating. In gametes that had been agglutinated by isolated mating type "minus" flagella, microfilaments and fertilization tubules progressively disassembled over a 3-h time course following flagellar disadhesion. Disassembly of microfilaments was inhibited by maintaining flagellar agglutination, suggesting that signals generated by flagellar adhesion were necessary to maintain microfilaments intact.