Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

<div><p>Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.</p></div

Redistribution of blastomeric Myo1C upon MyoI inhibition by PClP.

<p>(A) 3D rendered 100 μm cross section of Myo1C immunostaining profile in control 1 hpf embryo (inset- single confocal slice) (Bi) Normalized immunostaining intensity along the line drawn in Fig 4A inset, n = 5 embryos (Bi, top panel). Actin distribution at the same time, redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g003" target="_blank">Fig 3A</a>, left panel, n = 5 (Bi, middle panel). Membrane distribution profile perpendicular to surface of embryo shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g003" target="_blank">Fig 3D</a>, control)\, (Bi, bottom panel). (Bii) cartoon representation of Myo1C distribution in blastodisc cortex. (C) 3D rendered in 100 μm cross section of Myo1C immunostaining profile in 30 min PClP treated 1 hpf embryo, inset single confocal slice. (D) Comparative normalized intensity calculated long lines drawn in insets of (A&C)-slice views, control-cross, Myo1 inhibited-box, n = 5,. (E) Myo1C profiles for control 2 hpf, (F) Myo1 inhibited 2 hpf and (G) Normalized immunostaining intensity long lines drawn in insets of (E&F)-slice views, control (cross), Myo1 inhibited (box), n = 5, error bar indicates -standard deviation everywhere in Fig 4.</p

Comparison between role of Myo2 and Myo1, in furrow formation and LD dynamics.

<p>(A) Sideview, time-lapse profile of furrow maturation in control (top panel), Blebbistatin treated/Myo2 inhibited (middle panel) and PClP treated/Myo1 inhibited embryos (bottom panel). White dashed arrows in top panel and bottom panel indicate the formation of third furrow (marked as 3), parallel to the first furrow (marked as 1). Filled white arrows in bottom panel indicate the dissolving first-furrow and LD accumulation at that site, in Myo1 inhibited embryo. Filled white arrow in middle panel indicates dis-localization of second and third furrow. Bar 100 μm. (B) Top-view time-lapse profile of furrow maturation in control (top panel), Myo2 inhibited (middle panel) and Myo1 inhibited embryos (bottom panel). Arrows in bottom panel indicate LD accumulation along dissolving first furrow in Myo1 inhibited embryo. Bar 50 μm. (C) Cartoon diagram of embryo with (i) top and (ii) sideview position shown by eye symbol. (D) Scanning electron microscopy showing embryo blastomere surface of 64 cell control (top panel), equivalent time Myo2 inhibited (middle panel) and equivalent time Myo1 inhibited embryo (bottom panel). Bar 100 μM.</p

Inhibition of Myo1 arrests cell division and affected blastomere shapeof Zebrafish embryos.

<p>Schematic representation of Myo1 domain structures. (A) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Ea&b, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells stages for cDNA and RNA templates, (B) Semiquantitative RT-PCR profiles from cDNA and RNA templates. For Myo1Cb, control (top panel) and with PClP (bottom panel), were compared at 1–4 and 64 cells for cDNA and RNA templates. (C) (i) Western blot for Myo1C relative-levels at 1–4 and 64 cell stages. Control (top panel) and with PClP (bottom panel), GAPDH used as loading control. (ii) Relative change in levels of Myo1C ±PClP, 1–4 and 64 cell stages, control grey, PClP black. Error bars indicate SD, n = 3. (D) (i) Top panel, control embryos at different developmental stages. Bottom panel, PClP treated embryos taken in identical time as in control, dotted line shows boundary between yolk and blastodisc in both panels, (ii) measurement of changes in blastodisk thickness, as indicated by vertical both sided arrows in(Ei) with time, approximately along first cleavage furrow in both, the control (dotted line) and PClP treated embryos (solid line), n = 8, error indicate SD. Black arrow-head in time axis indicates PClP addition. Embryos were observed in lateral or side view position. (E) DAPI stained nucleus profile of 64-cell control (2 hpf) (top panel) and equivalent 2 hpf PClP inhibited 8- cell embryo (middle panel). 1 hpf control 8- cell embryo, showing divided nucleus is also shown (lower panel). bar 120μm in all places.</p

LDs gradually accumulate at the cleavage furrow of PClP treated embryo.

<p>(A-C) Lateral views of embryo, as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180301#pone.0180301.g002" target="_blank">Fig 2A</a>, Zoomed in on first cleavage furrow, (A) Control embryo from approximately 40 min post fertilization, montage of every 5 min, arrows indicates LDs near the first cleavage furrow. (B) Myo1 inhibited embryo from approximately 40 min post fertilization, montage of every 5 min. Arrows indicate accumulation of LDs at the first cleavage furrow line. (C) Myo2 inhibited embryo from approximately 40 min post fertilization, montage of every 5 min. Arrows indicate LDs near the first cleavage furrow. (D) Nile red staining of first cleavage furrow with lipid droplets shown by arrow in control embryo. (E) Nile red staining of first cleavage furrow with lipid droplet clump shown by arrow in Myo1 inhibited embryo. (F) Control, (G) Myo1 inhibited and (H) Myo2 inhibited embryos, kymograph view of average intensity along 10 μm line on both sides of the first cleavage furrow, ~ for approximately 40 min as shown in panels 5A-5C. Time is indicated along vertical axis and correlates with images in panel (5A-5C), approximate location of start and finish of 40 min time in kymographs are marked by arrows. LDs are indicated by black arrowheads. Scale bars are 50 μm in all images of this figure.</p

Pentabromopseudilin: a myosin V inhibitor suppresses TGF-<b>β</b> activity by recruiting the type II TGF-<b>β</b> receptor to lysosomal degradation

<p>Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria <i>Pseudomonas bromoutilis</i> and <i>Alteromonas luteoviolaceus</i>. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.</p

Methylation of the Fourth Position of Cholestanol Is Not Required for Dauer Larva Formation

<p>Structural formulae and space-filling models of (A) cholestanol, (B) lophanol, and (C) 4αF-cholestanol. Abilities to support reproductive growth or dauer formation in the second generation are indicated. R, reproduction; D, dauer larva.</p

Mutant <i>daf-16</i> Worms Grown on Lophenol Form Defective Dauer Larvae

<div><p>(A) Low-magnification electron micrograph of lophenol-grown <i>daf-16</i>. The alae are defective although the striated layer (bracket) is visible. Note that the gut is not constricted and contains remnants of food.</p> <p>(B and C) High-magnification electron micrographs of lophenol-grown <i>daf-16</i> and wild-type dauer larvae. Arrowhead indicates an annular structure.</p></div

Depletion of Cholesterol Is Associated with a Decrease of Nonmethylated Sterols

<div><p>(A) Nematode-specific biosynthesis of 4-methylated sterols from exogenously added cholesterol. Open arrow shows the methylation at the fourth position. A vertical line indicates hydrophilic metabolites of cholesterol.</p> <p>(B) Cholesterol metabolism in the first (lanes 1 and 2) and the second (lanes 3–6) generations of worms derived from mothers fed with radioactive cholesterol. CE, cholesteryl esters; mS, methylated sterols (lophenol, 4-methylcholestenol); nmS, nonmethylated sterols (cholesterol, 7-dehydrocholesterol, lathosterol). The position of these compounds on TLC was determined by chromatography of cholesteryl stearate, lophenol, and cholesterol. E, eggs; L1, L1 larvae.</p></div

Growth on Lophenol Induces the Accumulation of DAF-16 in the Nuclei of Neurons in a DAF-12–Dependent Manner

<div><p>(A) When grown on cholesterol, the transgenic line DAF-16a::GFP/b^KO displays a diffuse staining in the cytoplasm and nuclei of many cells (only the pharynx region of an L3 larva is shown).</p> <p>(B) Staining of a larva of similar age by Hoechst. Note many nuclei in the pharynx.</p> <p>(C) The DAF-16a::GFP/b^KO line grown on lophenol shows strong staining of nuclei in neurons of the pharynx, tail, and ventral cord of a dauer larva.</p> <p>(D) An L3 larva of DAF-16a::GFP/b^KO in a <i>daf-12</i> null background grown on lophenol. Note the diffuse fluorescence in the pharynx cell similar to that shown in (A).</p></div