Formins filter modified actin subunits during processive elongation☆
Introduction
Formins are a family of homodimeric proteins, each comprising two conserved domains in addition to more variable regulatory elements (Goode and Eck, 2007, Paul and Pollard, 2009b). Formin homology 2 (FH2) domains are composed of alpha-helices and connected head-to-tail to the partner FH2 domain by flexible linkers (Xu et al., 2004). These linkers can stretch enough for the FH2 dimer to wrap around an actin filament (Otomo et al., 2005), with a higher affinity for the barbed end than any other region of the filament. Formin homology 1 (FH1) domains are flexible polypeptides containing 2–12 polyproline sequences that bind profilin–actin complexes (Chang et al., 1997). Diffusion of the FH1 domains brings profilin–actin into contact with the barbed end (Kovar et al., 2006, Paul and Pollard, 2008), so that the complex can bind to the filament, followed by rapid dissociation of the profilin. The rate limiting step of formin mediated assembly of actin filaments is profilin–actin binding to the multiple FH1 sites, followed by the rapid transfer of actin onto the barbed end at a rate >1000 s−1 (Paul and Pollard, 2009a). As each new actin subunit adds to the barbed end, the formin steps reliably onto the new subunit, so formins move processively along growing barbed ends as more than 10,000 subunits are incorporated (Kovar et al., 2006, Paul and Pollard, 2008).
We are particularly interested in formin Cdc12p, which assembles actin filaments for the actomyosin contractile ring during cytokinesis in fission yeast. Fission yeast Schizosacchromyces pombe, is an excellent model organism to study the actin cytoskeleton, because powerful genetics, biochemistry and quantitative microscopy are available (for review see (Pollard and Wu, 2010)). These yeast cells have three structures composed of actin filaments: actin patches at sites of clathrin-mediated endocytosis, contractile rings for cytokinesis and actin cables for intracellular transport (for review, see (Kovar et al., 2011)). Genetics experiments showed that distinct proteins nucleate the actin filaments for these three structures: Arp2/3 complex nucleates filaments in actin patches (Morrell et al., 1999); formin Cdc12p nucleates filaments for the contractile ring (Chang et al., 1997); and formin For3p nucleates filaments for interphase cables (Feierbach and Chang, 2001). Deletion or temperature sensitive mutations of any of these actin nucleators leads to defects specifically in one type of actin structure but not the others. Each of these three nucleators localizes to distinct sites appropriate for the filaments they form in cells.
Although we know a great deal about the molecular components and assembly mechanism of contractile rings (Vavylonis et al., 2008, Wu and Pollard, 2005, Wu et al., 2003), we still know very little about the dynamics of actin filaments during the assembly and disassembly of the rings. This information would greatly enhance our understanding of the mechanism of cytokinesis. However, such an effort had been hampered by the lack of a fluorescence probe of actin filaments. Formins (Bni1, mDia1, mDia2, Cdc12p) incorporate actin subunits labeled with Oregon green (molecular weight 463) on cys374 or Alexa green (molecular weight 643) on a lysine side chain into actin filaments in biochemical assays (Kovar and Pollard, 2004). However, actin with an N-terminal fluorescent protein tag does not incorporate into the actin filaments of the contractile ring in fission yeast (Wu and Pollard, 2005). Since these actin filaments are essential for cell division, replacement of the native actin gene with a gene encoding a fusion of a fluorescent protein and actin results in the failure of cytokinesis and the cells die, apparently because formin Cdc12p rejects all of the fusion protein. Although fission yeast cells cannot survive on GFP–actin alone, they tolerate low levels of expression of GFP–actin in the presence of wild type levels of native actin (Wu and Pollard, 2005). These trace levels of GFP–actin can be used to make quantitative measurements of actin filaments in actin patches (Sirotkin et al., 2010) but not contractile rings.
Although probes such as fluorescent protein-tagged calponin homology domains (CHD) (Wachtler et al., 2003) and Lifeact (Riedl et al., 2008) can be used to label actin filaments indirectly in vivo, they have shortcomings. These probes bind actin filaments with Kds of about 2–50 μM (Gimona et al., 2002, Mateer et al., 2004, Riedl et al., 2008), thus they must be expressed at a relatively high concentration to detect actin filaments in cells, which could perturb the actin cytoskeleton. More importantly, none of these indirect fluorescent probes can be used for quantitative fluorescence microscopy to characterize the dynamics of actin filaments in live cells, which requires direct labeling of the protein being studied (Wu and Pollard, 2005). Here we inserted a tetracysteine peptide at 10 sites in fission yeast actin, and labeled these proteins with the FlAsH reagent in live cells (Adams et al., 2002, Griffin et al., 1998). The combined size of the peptide and dye is less than 2 kDa. Some tetracysteine tagged actins were incorporated into actin filaments nucleated by Arp2/3 complex in actin patches, but to our surprise formin Cdc12p excluded all of these labeled actin molecules from contractile rings. Here we explore the structural basis for this rejection of labeled actin by formins.
Section snippets
Mutagenesis and expression of mutant actins
Fission yeast strains with inserts in the actin gene were made by site directed mutagenesis. The strains used in this study are listed in Table 2. We subcloned the S. pombe act1+ cDNA into pFA6a-KanMX6 to make pFA6a-Act-KanMX6. Then we inserted the coding sequence for a peptide with a tetracysteine motif (FLNCCPGCCMEP) (Martin et al., 2005) into the actin coding sequence by site-directed mutagenesis (QuickChange II, Stratagene, CA) and used the plasmid to replace the ura4+ cassette in the
Mutagenesis strategy
To create sites in actin for labeling with the FlAsH reagent in live cells, we inserted the coding sequence for a dodecapeptide containing a tetracysteine motif (FLNCCPGCCMEP) at either the N- or C-terminus or at one of eight different internal sites in the fission yeast actin gene (Table 1, Table 2). All of the internal sites were in surface loops including three in the DNase binding loop (Fig. 1A). We avoided the nucleotide binding cleft and sites where actin is known to interact with other
Discussion
We added tetracysteine tags at eight internal sites in fission yeast actin in addition to the N- and C-termini. The FlASH dye labeled most of these mutant proteins in live cells. Six of the actins with inserted tags and actin with the C-terminal tag were not incorporated into any actin structure. Models of the actin filament (Oda et al., 2009) (Fig. 1C) showed that inserts at these six sites are likely to interfere with actin polymerization, so they were excluded from all the filaments. The
Conclusions
Fission yeast cells incorporated three different FlAsH-actin constructs to filaments nucleated by Arp2/3 complex at sites of endocytosis but not into the contractile ring. Therefore, Cdc12p, the fission yeast formin required for the assembly of contractile rings, has very stringent structural requirements for incorporating subunits at actin filament barbed ends as it moves processively along the end of a growing filament.
Acknowledgments
This work was supported by NIH research Grants GM026132 and GM-026338. The authors thank Vladimir Sirotkin and Naomi Courtemanche for helpful discussion.
References (41)
- et al.
Structural memory in the contractile ring makes the duration of cytokinesis independent of cell size
Cell
(2009) - et al.
Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division
Curr. Biol.
(2001) - et al.
Cytokinesis without myosin II
Curr. Opin. Cell Biol.
(2000) - et al.
Functional plasticity of CH domains
FEBS Lett.
(2002) - et al.
Three’s company: the fission yeast actin cytoskeleton
Trends Cell Biol.
(2011) - et al.
Control of the assembly of ATP- and ADP-actin by formins and profilin
Cell
(2006) - et al.
Structure of the FH2 domain of Daam1: implications for formin regulation of actin assembly
J. Mol. Biol.
(2007) - et al.
Dynamics of the formin for3p in actin cable assembly
Curr. Biol.
(2006) - et al.
Myosin-II-dependent localization and dynamics of F-actin during cytokinesis
Curr. Biol.
(2005) - et al.
The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation
Curr. Biol.
(2008)
Energetic requirements for processive elongation of actin filaments by FH1FH2-formins
J. Biol. Chem.
Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis
Dev. Cell
Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture
Cell
New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications
J. Am. Chem. Soc.
Structure of crystalline actin sheets
Nature
A green fluorescent protein-actin fusion protein dominantly inhibits cytokinesis, cell spreading, and locomotion in Dictyostelium
Cell Struct. Funct.
The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling
Bioinformatics
Role of polo kinase and Mid1p in determining the site of cell division in fission yeast
J. Cell Biol.
Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle
Mol. Biol. Cell
Cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin
J. Cell Biol.
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Dedication: On the occasion of Ueli Aebi’s retirement, I am happy to share some of our recent work on Ueli’s second most favorite protein, actin. Ueli and I enjoyed working together on actin in the early 1980s at the Johns Hopkins Medical School. With Gerhard Isenberg we discovered capping protein (Isenberg et al., 1980) and with Ross Smith determined an excellent low resolution structure of the actin molecule based on three-dimensional reconstructions from electron micrographs of two-dimensional crystals of actin (Aebi et al., 1980). Rather than attempting in this paper a comprehensive review of our recent work on the role of actin in cytokinesis and cellular motility, we will describe one new result regarding the structural requirements for formins to elongated actin filaments. Thomas D. Pollard, New Haven, CT, USA, 2011.