Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Abstract

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using "bulk" and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by "trapping" Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast

“…Inserts were sequenced to confirm fidelity of PCR amplification. The fission yeast tropomyosin Cdc8 expression plasmid pREP4x-cdc8 has been described (29). The bacterial expression plasmid for cofilin Adf1 (pMW-SpCofilin) and GSTWsp1 (pGEX2-(SpWsp1p)VCA) have been described (3,12).…”

“…Protein Purification-Tropomyosin (Cdc8) was purified from fission yeast cells expressing pREP4X-cdc8 by successive steps of boiling, ammonium sulfate precipitation, and ion-exchange chromatography as described (29). Full-length recombinant fission yeast fimbrin Fim1 and GST-Wsp1 were purified as previously described (3,23).…”

“…Gel-filtered actin was labeled on Cys-374 with pyrenyl iodoacetamide or Oregon Green iodoacetamide (Invitrogen) (26 Actin Filament Sedimentation-Actin filaments preassembled for 2 h at 25°C from 15 M Mg-ATP-actin monomers were incubated with a range of concentrations of Fim1 for 20 min at 25°C and then spun at 100,000 ϫ g (high speed) or 10,000 ϫ g (low speed) for 20 min at 25°C. Equal volumes of total (before centrifugation), supernatant, and pellet were separated by 12.5% SDS-polyacrylamide gel electrophoresis, stained with Coomassie Blue for 30 min, and destained for 16 h. Gels were analyzed by densitometry on an Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NE) as previously described (29). The amount of protein was quantified as depletion from the supernatant, converted to micromolar bound.…”

Tropomyosin is a tetramer under physiological salt conditions

“…Members of the formin family of proteins act as processive polymerases incorporating actin from P:A whilst remaining bound at the (þ)-end of the filaments [Carlier and Pantaloni, 2007;Renault et al, 2008]. Recently, the function of tropomyosin (TM), in the regulation of the actin filament (þ)-end has received increased attention [Hillberg et al, 2006;Wawro et al, 2007;Grenklo et al, 2008;Bach et al, 2009;Skau et al, 2009].Classically, TM participates in the calcium control of actomyosin force generation in the muscle, yet it is an abundant multi-isoform protein in non-muscle cells, see [Gunning et al, 2005. The isoforms are classified into two groups based on their polypeptide length; the low molecular weight (LMW; 247 amino acid residues and mw 28,000), and the high molecular weight (HMW; 284 residues and 32,000) isoforms.…”

Regulation and function of the fission yeast myosins

“…Myo2 first appears at the division site as dots that colocalise with the fission yeast anillin-like protein Mid1 (Wong et al, 2002). At the onset of anaphase, Myo2 then potentially interacts with formin-associated actin seeds and tropomyosin to complete formation of the CAR (Mulvihill and Hyams, 2002;Skau et al, 2009;Stark et al, 2010). Once the CAR is formed, Myo2 can be rapidly exchanged (Wong et al, 2002), a process that is controlled by the conserved UCSdomain-containing protein Rng3, which is required for ensuring CAR integrity (Lord and Pollard, 2004;Lord et al, 2008;Wong et al, 2000).…”