Spire2 and Rab11a synergistically activate myosin-5b motor function.

Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles.

Insect Sf9 cells are suitable for functional expression of insect, but not vertebrate, striated muscle myosin.

Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.

The cargo adaptor proteins RILPL2 and melanophilin co-regulate myosin-5a motor activity.

Vertebrate myosin-5a is an ATP-utilizing processive motor associated with the actin network and responsible for the transport and localization of several vesicle cargoes. To transport cargo efficiently and prevent futile ATP hydrolysis, myosin-5a motor function must be tightly regulated. The globular tail domain (GTD) of myosin-5a not only functions as the inhibitory domain but also serves as the binding site for a number of cargo adaptor proteins, including melanophilin (Mlph) and Rab-interacting lysosomal protein-like 2 (RILPL2). In this study, using various biochemical approaches, including ATPase, single-molecule motility, GST pulldown assays, and analytical ultracentrifugation, we demonstrate that the binding of both Mlph and RILPL2 to the GTD of myosin-5a is required for the activation of myosin-5a motor function under physiological ionic conditions. We also found that this activation is regulated by the small GTPase Rab36, a binding partner of RILPL2. In summary, our results indicate that RILPL2 is required for Mlph-mediated activation of Myo5a motor activity under physiological conditions and that Rab36 promotes this activation. We propose that Rab36 stimulates RILPL2 to interact with the myosin-5a GTD; this interaction then induces exposure of the Mlph-binding site in the GTD, enabling Mlph to interact with the GTD and activate myosin-5a motor activity.

Regulation of Myosin-5b by Rab11a and the Rab11 family interacting protein 2.

Mammalian myosin-5b (Myo5b) plays a critical role in the recycling of endosomes to the plasma membrane via the interactions with Rab11a and the Rab11 family interacting protein 2 (FIP2). However, it remains unclear on how Rab11a and FIP2 are coordinated in tethering Myo5b with the vesicles and activating the motor function of Myo5b. In the present study, we show that Rab11a binds to the globular tail domain (GTD) of Myo5b and this binding abolishes the head-GTD interaction of Myo5b, thus activating the motor function of Myo5b. On the other hand, FIP2 directly interacts with both Rab11a and the tail of Myo5b, and the binding of FIP2 to Myo5b does not affect Myo5b motor function. Moreover, Rab11a displays higher affinity to FIP2 than to Myo5b, suggesting that Rab11a binds preferentially to FIP2 than to Myo5b. Based on the current findings, we propose that the association of Myo5b with vesicles is mediated by FIP2, which bridges Myo5b and the membrane-bound Rab11a, whereas the motor function of Myo5b is regulated by Rab11a.

Regulation of class V myosin.

Class V myosin (myosin-5) is a molecular motor that functions as an organelle transporter. The activation of myosin-5's motor function has long been known to be associated with a transition from the folded conformation in the off-state to the extended conformation in the on-state, but only recently have we begun to understand the underlying mechanism. The globular tail domain (GTD) of myosin-5 has been identified as the inhibitory domain and has recently been shown to function as a dimer in regulating the motor function. The folded off-state of myosin-5 is stabilized by multiple intramolecular interactions, including head-GTD interactions, GTD-GTD interactions, and interactions between the GTD and the C-terminus of the first coiled-coil segment. Any cellular factor that affects these intramolecular interactions and thus the stability of the folded conformation of myosin-5 would be expected to regulate myosin-5 motor function. Both the adaptor proteins of myosin-5 and Ca2+ are potential regulators of myosin-5 motor function, because they can destabilize its folded conformation. A combination of these regulators provides a versatile scheme in regulating myosin-5 motor function in the cell.

Melanophilin Stimulates Myosin-5a Motor Function by Allosterically Inhibiting the Interaction between the Head and Tail of Myosin-5a.

The tail-inhibition model is generally accepted for the regulation of myosin-5a motor function. Inhibited myosin-5a is in a folded conformation in which its globular tail domain (GTD) interacts with its head and inhibits its motor function, and high Ca(2+) or cargo binding may reduce the interaction between the GTD and the head of myosin-5a, thus activating motor activity. Although it is well established that myosin-5a motor function is regulated by Ca(2+), little is known about the effects of cargo binding. We previously reported that melanophilin (Mlph), a myosin-5a cargo-binding protein, is capable of activating myosin-5a motor function. Here, we report that Mlph-GTBDP, a 26 amino-acid-long peptide of Mlph, is sufficient for activating myosin-5a motor function. We demonstrate that Mlph-GTBDP abolishes the interaction between the head and GTD of myosin-5a, thereby inducing a folded-to-extended conformation transition for myosin-5a and activating its motor function. Mutagenesis of the GTD shows that the GTD uses two distinct, non-overlapping regions to interact with Mlph-GTBDP and the head of myosin-5a. We propose that the GTD is an allosteric protein and that Mlph allosterically inhibits the interaction between the GTD and head of myosin-5a, thereby activating myosin-5a motor function.

The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11.

In the Drosophila melanogaster compound eye, myosin-5 (DmM5) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. However, little is known of how the motor function of DmM5 is regulated at the molecular level. In the present study, we overexpressed DmM5 in Sf9 insect cells and investigated its regulation using purified proteins. We found that the actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid (Ltd) and dRab11 respectively. We found that GTP-bound dRab11, but not Ltd, significantly activates DmM5 actin-activated ATPase activity. Moreover, we identified Gln(1689) in the GTD as the critical residue for the interaction with dRab11 and activation of DmM5 motor function by dRab11. Based on those results, we propose that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.

[Pollution characteristics and distribution of polycyclic aromatic hydrocarbons and organochlorine pesticides in groundwater at Xiaodian Sewage Irrigation Area, Taiyuan City].

Sewage irrigation has been widely used in areas of water shortage in northern China, and it may introduce organic contaminants into groundwater. To characterize the organic contaminants in groundwater in sewage irrigation area, the Xiaodian sewage irrigation area in Shanxi Province was chosen as the case study area. A total of 16 groundwater samples (13 from shallow aquifer, 3 from deep aquifer) were collected. Polycyclic aromatic hydrocarbons (PAHs) were determined by gas chromatography-mass spectrometry (GC-MS) and organochlorine pesticides (OCPs) were ainalyzed by gas chromatography-electron capture detection (GC-ECD). The results showed that the concentrations of PAHs ranged from 13.98 to 505.89 ng x L(-1) with an average concentration of 115.67 ng x (L)(-1). The 2 and 3 ring-PAHs were the main components, while naphthalene and phenanthrene were most frequently detected. The concentrations of OCPs were in the range of 13.91-103.23 ng x L(-1) with an average concentration of 40.99 ng x L(-1), while alpha-HCH, delta-HCH, o,p'-DDD, Aldrin, Endosulfan-sulfate and HCB were most frequently detected. Overall, shallow aquifers appeared more contaminated with these pollutants than deep aquifers. In the area, the order of the organic contaminants concentration in groundwater was: East Main Channel < Beizhang Drainage < Taiyu Drainage, which indicated the quality of groundwater was influenced by the sewage irrigation.

[Influence of organochlorine pesticides in wastewater on the soil along the channel].

Nine profile soil samples and two sewage water samples were collected from Xiaodian sewage irrigation area in Taiyuan city, concentrations of organochlorine pesticides (OCPs) were determined by the gas chromatography coupled with electron capture detector (GC-ECD) to analyze the influence of the leakage of sewage water. The result shows that OCPs in sewage water were mainly composed of HCHs. Concentrations of DDTs and other organochlorine pesticides were very low or out of the detection limit. Concentrations of sigmaOCPs and HCHs in eight profiles near irrigation channels to some extend decreased with the increasing of the linear distance off the channel, which shows influences of the leakage of sewage water on the soil nearby. Concentrations of HCHs clearly decreased with the increasing of soil depth in most profile soils. For the horizontal direction, concentrations of HCHs also decreased with the increasing of the linear distance off the channel. The correlation between HCHs and TOC was positive, but no correlation between pH and HCHs was found.

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a.

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.