Thomas E. Sladewski

Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

A combination of in vivo and in vitro approaches were used to show how tropomyosin and myosin-II contribute to contractile ring assembly in fission yeast. Ring assembly is sensitive to changes in the cellular levels of myosin-II, and tropomyosin works to maximize myosin-II motor function during this process by stabilizing actomyosin interactions.

Tropomyosin Is Essential for Processive Movement of a Class V Myosin from Budding Yeast

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class V myosins were characterized as nonprocessive in vitro, including Myo2p, the essential class V myosin from S. cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or behave processively in the cellular context. Here we show that Myo2p moves processively in vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.

Yeast UCS proteins promote actomyosin interactions and limit myosin turnover in cells

Two functions are proposed for the conserved family of UCS proteins: helping to fold myosin motor proteins and stimulating the motor function of folded myosins. We examined both functions in yeast. The fission yeast UCS protein (Rng3p) concentrates in nodes containing myosin-II (Myo2) and other proteins that condense into the cytokinetic contractile ring. Both the N-terminal (central) and C-terminal (UCS) domains of Rng3p can concentrate independently in contractile rings, but only full-length Rng3p supports contractile ring function in vivo. The presence of Rng3p in ATPase assays doubles the apparent affinity (KATPase) of both native Myo2 and recombinant heads of Myo2 for actin filaments. Rng3p promotes gliding of actin filaments by full-length Myo2 molecules, but not Myo2 heads alone. Myo2 isolated from mutant strains defective for Rng3p function is soluble and supports actin filament gliding. In budding yeast the single UCS protein (She4p) acts on both myosin-I isoforms (Myo3p and Myo5p) and one of two myosin-V isoforms (Myo4p). Myo5p turns over ≈10 times faster in she4Δ cells than wild-type cells, reducing the level of Myo5p in cells 10-fold and in cortical actin patches ≈4-fold. Nevertheless, Myo5p isolated from she4Δ cells has wild-type ATPase and motility activities. Thus, a fraction of this yeast myosin can fold de novo in the absence of UCS proteins, but UCS proteins promote myosin stability and interactions with actin.

Two single-headed myosin V motors bound to a tetrameric adapter protein form a processive complex

The yeast class V myosin Myo4p moves processively in vivo in a cargo-dependent manner following formation of a double-headed complex with the adapter protein She3p and the mRNA-binding protein She2p.

Regulation of Fission Yeast Myosin-II Function and Contractile Ring Dynamics by Regulatory Light-Chain and Heavy-Chain Phosphorylation

We investigated the role of regulatory light-chain (Rlc1p) and heavy-chain phosphorylation in controlling fission yeast myosin-II (Myo2p) motor activity and function during cytokinesis. Phosphorylation of Rlc1p leads to a fourfold increase in Myo2ps in vitro motility rate, which ensures effective contractile ring constriction and function. Surprisingly, unlike with smooth muscle and nonmuscle myosin-II, RLC phosphorylation does not influence the actin-activated ATPase activity of Myo2p. A truncated form of Rlc1p lacking its extended N-terminal regulatory region (including phosphorylation sites) supported maximal Myo2p in vitro motility rates and normal contractile ring function. Thus, the unphosphorylated N-terminal extension of Rlc1p can uncouple the ATPase and motility activities of Myo2p. We confirmed the identity of one out of two putative heavy-chain phosphorylation sites previously reported to control Myo2p function and cytokinesis. Although in vitro studies indicated that phosphorylation at Ser-1444 is not needed for Myo2p motor activity, phosphorylation at this site promotes the initiation of contractile ring constriction.

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase.

Fission yeast myosin-I (Myo1p) not only associates with calmodulin, but also employs a second light chain called Cam2p. cam2Δ cells exhibit defects in cell polarity and growth consistent with a loss of Myo1p function. Loss of Cam2p leads to a reduction in Myo1p levels at endocytic patches and a 50% drop in the rates of Myo1p-driven actin filament motility. Thus, Cam2p plays a significant role in Myo1p function. However, further studies indicated the existence of an additional Cam2p-binding partner. Cam2p was still present at cortical patches in myo1Δ cells (or in myo1-IQ2 mutants, which lack an intact Cam2p-binding motif), whereas a cam2 null (cam2Δ) suppressed cytokinesis defects of an essential light chain (ELC) mutant known to be impaired in binding to PI 4-kinase (Pik1p). Binding studies revealed that Cam2p and the ELC compete for Pik1p. Cortical localization of Cam2p in the myo1Δ background relied on its association with Pik1p, whereas overexpression studies indicated that Cam2p, in turn, contributes to Pik1p function. The fact that the Myo1p-associated defects of a cam2Δ mutant are more potent than those of a myo1-IQ2 mutant suggests that myosin light chains can contribute to actomyosin function both directly and indirectly (via phospholipid synthesis at sites of polarized growth).

A single molecule approach to mRNA transport by a class V myosin

mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. We show that an in vitro single molecule approach, using purified recombinant full-length proteins and synthesized mRNA, provides insight into the mechanism by which localizing mRNAs are carried to their destination. A messenger ribonucleoprotein (mRNP) complex was reconstituted from a budding yeast class V myosin motor complex (Myo4p–She3p), an mRNA-binding adaptor protein (She2p), and a localizing mRNA (ASH1). The motion of the mRNP was tracked with high spatial (∼10 nm) and temporal (70 ms) resolution. Using this “bottom-up” methodology, we show that mRNA triggers the assembly of a high affinity double-headed motor-mRNA complex that moves continuously for long distances on actin filaments at physiologic ionic strength. Without mRNA, the myosin is monomeric and unable to move continuously on actin. This finding reveals an elegant strategy to ensure that only cargo-bound motors are activated for transport. Increasing the number of localization elements (“zip codes”) in the mRNA enhanced both the frequency of motile events and their run length, features which likely enhance cellular localization. Future in vitro reconstitution of mRNPs with kinesin and dynein motors should similarly yield mechanistic insight into mRNA transport by microtubule-based motors.

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex

We investigated the role of full-length Drosophila Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and K10 mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one K10 mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.