title: Evidence That an Unconventional Actin Can Provide Essential F-Actin Function and That a Surveillance System Monitors F-Actin Integrity in Chlamydomonas.
abstract: Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Onishi
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 jpringle@stanford.edu fcross@mail.rockefeller.edu.
authors.forename: John R
authors.initials: JR
authors.lastname: Pringle
authors.affiliation: The Rockefeller University, New York, New York 10065 jpringle@stanford.edu fcross@mail.rockefeller.edu.
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Cross
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000378:metabolism; D000072020:Synthetic Lethal Mutations; D053778:Thiazolidines / Q000737:chemistry; D015854:Up-Regulation
chemical_list: D000199:Actins; D020418:Algal Proteins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D053778:Thiazolidines; C037068:latrunculin B
keywords: Chlamydomonas; actin; algal and plant cytoskeletons; latrunculin; mutation identification by sequencing