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Genetics. 2016 Mar;202(3):977-96. doi: 10.1534/genetics.115.184663. Epub 2015 Dec 29.

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

Author information

1
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305.
2
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305 jpringle@stanford.edu fcross@mail.rockefeller.edu.
3
The Rockefeller University, New York, New York 10065 jpringle@stanford.edu fcross@mail.rockefeller.edu.

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.

KEYWORDS:

Chlamydomonas; actin; algal and plant cytoskeletons; latrunculin; mutation identification by sequencing

PMID:
26715672
PMCID:
PMC4788133
[Available on 2017-03-01]
DOI:
10.1534/genetics.115.184663
[Indexed for MEDLINE]
Free PMC Article

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