Abstract
In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.
Highlights
Cell-cell interactions leading to fusion between gametes of opposite sexes during fertilization are complex processes that involve dramatic changes in each of the interacting gametes
Because many of the cellular events that comprise gamete activation can be induced by incubation of gametes of a single mating type in dibutyryl cAMP, we investigated the influence of this molecule on Chlamydomonas aurora/Ipl1p-like protein kinase (CALK)
Dibutyryl cAMP induced movement of CALK to the flagella. These results indicated that flagellar adhesion per se was not required for translocation and showed that CALK translocation is a part of gamete activation
Summary
Cell-cell interactions leading to fusion between gametes of opposite sexes during fertilization are complex processes that involve dramatic changes in each of the interacting gametes. Interactions between adhesion molecules/receptors on the sperm plasma membrane and ligands on the egg surface activate poorly understood signaling. At the completion of gametogenesis, during which asexually growing mtϩ and mtϪ vegetative cells differentiate into sexually competent cells, the resulting mtϩ and mtϪ gametes bear mating type-specific adhesion molecules, agglutinins, on their flagellar membranes and on the plasma membrane of the cell body. The agglutinins on the flagella are in an active state and are capable of binding to flagellar agglutinins on gametes of the opposite mating type, whereas agglutinin molecules on the contiguous plasma membrane of the cell body are inactive [6]. In addition to binding the flagella of gametes of opposite mating types to each other, these receptor/ligand-like interactions initiate a complex series of events termed gamete activation. As part of an intricate feedback mechanism required to maintain and enhance flagellar adhesiveness and to keep the cells bound to each other until cell-cell fusion occurs, cAMP levels increase in the cell bodies of the gametes of both mating types during flagellar adhesion and lead to increased flagellar adhesiveness [6, 9, 12, 13]
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