Expression of Conventional and Unconventional Actins in Chlamydomonas reinhardtii upon Deflagellation and Sexual Adhesion

Abstract

Chlamydomonas has two actin genes, one coding for a conventional actin and the other coding for a highly divergent actin. The divergent actin NAP (for "novel actin-like protein") is expressed only negligibly in wild-type cells but abundantly in a null mutant of conventional actin, the ida5 mutant. The presence of the dormant NAP gene suggests that NAP may also have its own function in wild-type cells under some conditions. However, no specific functions have been suggested. In this study, we examined the expression of actin and NAP in wild-type and ida5 cells under conditions where actin function has been shown to be important. We found that deflagellation induces the expression of NAP as well as that of actin in wild-type cells. The expressed NAP becomes localized to the regrown flagella, apparently without being associated with dynein. Mating of gametes also increased the expression of actin in wild-type cells and that of NAP in ida5 cells, resulting in accumulation of these proteins in flagella (in both wild-type and ida5 cells) and the fertilization tubule (only in wild-type cells). However, it did not induce significant NAP expression in wild-type cells. These and other observations suggest that the expression of actin and NAP mRNAs is controlled by two discrete mechanisms and that NAP plays a role in flagellar formation in wild-type cells.

Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity inChlamydomonas

“…The normal proliferation of the ida5-1 null mutant (KatoMinoura et al 1997), upregulation of the highly divergent actin NAP1 in that mutant (Kato-Minoura et al 1998), and uncertainty both about the presence of F-actin in vegetative cells (Harper et al 1992;Kato-Minoura et al 1998) and about the polymerization ability of NAP1 (Hirono et al 2003;KatoMinoura 2011) raised questions that the isolation of the nap1 and lat mutants have now allowed us to answer, revealing some important and rather surprising aspects of actin structure and function. First, the inviability of the nap1, lat1, lat2, and lat3 mutants either in combination with ida5-1 or upon treatment with LatB appears to demonstrate both that actin function is essential in vegetative Chlamydomonas cells and that either the conventional actin IDA5 or the highly divergent actin NAP1 can provide the essential function(s).…”

“…Although NAP1 is only $65% identical in amino acid sequence to IDA5 and other conventional actins ( Figure 1B), phylogenetic analyses consistently cluster it together with actins rather than with Arp1-type actin-related proteins ( Figure 1A; Kato-Minoura et al 2014). NAP1 is transcriptionally upregulated in the ida5-1 null mutant (Kato-Minoura et al 1998;Hirono et al 2003), so that it has been speculated that it could provide actin function under these conditions. A priori, the extensive sequence divergence of NAP1 from conventional actins provides a challenge for this model.…”

“…It has a single gene (IDA5) encoding a conventional actin (Figure 1,A and B), and localization studies have suggested that IDA5 may have multiple roles both during vegetative growth and during mating (Harper et al 1992;Ehler and Dutcher 1998;Kato-Minoura et al 1998;Kovar et al 2001;Avasthi et al 2014). However, electron microscopy and labeling with fluorescent phallotoxin detected F-actin structures only in fertilization tubules (Detmers et al 1983;Wilson et al 1997;Hirono et al 2003), raising the surprising possibility that IDA5 might function during vegetative growth as G-actin rather than in filaments, as originally suggested by Harper et al (1992). In another surprise, a screen for mutants defective in swimming behavior and flagellar structure yielded a null mutation in IDA5 (originally ida5, henceforth ida5-1; a frameshift resulting in a complete loss of IDA5 protein) (Kato et al 1993;Kato-Minoura et al 1997).…”

Immunolocalization of Actin in Paramecium Cells

“…This applies to early steps of exocytosis, including dense core vesicle docking (Morales et al 2000;Pendleton and Koffer 2001;Manneville et al 2003;Gasman et al 2004), late steps of endocytosis (Engqvist-Goldstein and Drubin 2003;Guilherme et al 2004), exo-endocytosis coupling (Valentijn et al 1999), endo-phagosome interaction (Kjeken et al 2004), delivery of endocytosed receptors to lysosomes for degradation (Stoorvogel et al 2004), vacuole fusion in yeast (Merz and Wickner 2004), and positioning of the nucleus (Starr and Han 2003). Some aspects are still poorly understood, particularly, e.g., the role of actin in flagella of algae (Mitchell 2000;Hayashi et al 2001;Hirono et al 2003), whereas its occurrence in cilia has remained a matter of debate. Another line of experiments concerns the potential role of actin in mediating the connection between cortical Ca 2 ϩ -stores and the plasma membrane (Patterson et al 1999;Rosado and Sage 2000;Kunzelmann-Marche et al 2001;Wang et al 2002).…”

Impaired Flagellar Regeneration Due to Uncoordinated Expression of Two Divergent Actin Genes in Chlamydomonas

“…This discovery has caused us to wonder why the NAP gene is preserved in wild-type cells, which express only negligible amounts of NAP under normal conditions. Recently, however, Hirono et al (2003) found that NAP is expressed shortly after flagellar amputation in wild-type cells. From this observation, these investigators suggested that NAP might somehow be involved in the flagellation process.…”