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.

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation identification (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely configured process in kinetoplastids.

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.

We investigated the role of binding partners of full-length Drosophila Bicaudal D (BicD) in the activation of dynein-dynactin motility for mRNA transport on microtubules. In single-molecule assays, full-length BicD robustly activated dynein-dynactin only when both the mRNA binding protein Egalitarian (Egl), and K10 mRNA cargo were present. Electron microscopy showed that both Egl and mRNA were needed to disrupt an auto-inhibited, looped BicD conformation that sterically prevents dynein-dynactin binding. In vitro reconstituted messenger ribonucleoprotein (mRNP) complexes with two Egl molecules showed faster speeds and longer run lengths than mRNPs with one Egl, suggesting that cargo binding enhances dynein recruitment. Labeled dynein showed that BicD can recruit two dimeric dyneins to the mRNP, resulting in faster speeds and longer run lengths than with one dynein. The fully reconstituted mRNP provides a model for understanding how adaptor proteins and cargo cooperate to confer optimal transport properties to a dynein-driven transport complex.

Molecular motors are instrumental in mRNA localization, which provides spatial and temporal control of protein expression and function. To obtain mechanistic insight into how a class V myosin transports mRNA, we performed single-molecule in vitro assays on messenger ribonucleoprotein (mRNP) complexes that were reconstituted from purified proteins and a localizing mRNA found in budding yeast. mRNA is required to obtain a stable processive transport complex on actin, an elegant mechanism to ensure that only cargo-bound motors are motile. Increasing the number of localizing elements (“zipcodes”) on the mRNA, or configuring the track to resemble actin cables, enhanced run length and event frequency. In multi-zipcode mRNPs, motor separation distance varied during a run, showing the dynamic nature of the transport complex. Building the complexity of single-molecule in vitro assays is necessary to understand how these complexes function within cells

Summary 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.

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 Myo2p's 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.

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.