Multivariate analysis of genomic variables, effective population size, and mutation rate

Abstract Objective The relationship between genomic variables (genome size, gene number, intron size, and intron number) and evolutionary forces has two implications. First, they help to unravel the mechanism underlying genome evolution. Second, they provide a solution to the debate over discrepancy between genome size variation and organismal complexity. Previously, a clear correlation between genomic variables and effective population size and mutation rate (Neu) led to an important hypothesis to consider random genetic drift as a major evolutionary force during evolution of genome size and complexity. But recent reports also support natural selection as the leading evolutionary force. As such, the debate remains unresolved. Results Here, we used a multivariate method to explore the relationship between genomic variables and Neu in order to understand the evolution of genome. Previously reported patterns between genomic variables and Neu were not observed in our multivariate study. We found only one association between intron number and Neu, but no relationships were observed between genome size, intron size, gene number, and Neu, suggesting that Neu of the organisms solely does not influence genome evolution. We, therefore, concluded that Neu influences intron evolution, while it may not be the only force that provides mechanistic insights into genome evolution and complexity

Ascidian caveolin induces membrane curvature and protects tissue integrity and morphology during embryogenesis

Cell morphology and tissue integrity are essential for embryogenesis. Caveolins are membrane proteins that induce the formation of surface pits called caveolae that serve as membrane reservoirs for cell and tissue protection during development. In vertebrates, caveolin 1 (Cav1) and caveolin 3 (Cav3) are required for caveola formation. However, the formation of caveola and the function of caveolins in invertebrates are largely unknown. In this study, three caveolins, Cav-a, Cav-b, and CavY, are identified in the genome of the invertebrate chordate Ciona spp. Based on phylogenetic analysis, Cav-a is found to be closely related to the vertebrate Cav1 and Cav3. In situ hybridization shows that Cav-a is expressed in Ciona embryonic notochord and muscle. Cell-free experiments, model cell culture systems, and in vivo experiments demonstrate that Ciona Cav-a has the ability to induce membrane curvature at the plasma membrane. Knockdown of Cav-a in Ciona embryos causes loss of invaginations in the plasma membrane and results in the failure of notochord elongation and lumenogenesis. Expression of a dominant-negative Cav-a point mutation causes cells to change shape and become displaced from the muscle and notochord to disrupt tissue integrity. Furthermore, we demonstrate that Cav-a vesicles show polarized trafficking and localize at the luminal membrane during notochord lumenogenesis. Taken together, these results show that the invertebrate chordate caveolin from Ciona plays crucial roles in tissue integrity and morphology by inducing membrane curvature and intracellular vesicle trafficking during embryogenesis

An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division

<div><p>Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate <i>Ciona intestinalis</i>, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation.</p></div

Cortical flow contributes to the formation of the equatorial actomyosin ring.

<p>(A) Time-lapse projections of a notochord cell showing that lifeact-mEGFP–labeled actin filaments (white and yellow arrowheads follow two circumferential actin filaments) move toward and align parallel to the equator in the equatorial region (bracket) (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001781#pbio.1001781.s014" target="_blank">Movie S3</a>). In addition to the circumferential long actin filaments, between the lateral domain and the equatorial region, a population of longitudinally oriented short filaments (green arrowhead follows a single filament) move into the equatorial region, where they join the circumferential filaments. (B) Dynamic movement of mCherry-MRLC–labeled myosin filaments toward the equator in the equatorial region (bracket). White arrowhead follows a myosin filament. (C) Confocal section (1 µm) of a notochord cell double labeled for actin (lifeact-mEGFP) and myosin (mCherry-MRLC) shows that actin filaments and myosin filaments are present simultaneously in the contractile ring. (D) Colocalization analysis of the equatorial region (box in C) shows that a significant amount of actin filaments and myosin filaments do not colocalize. (E) Manual tracking of single actin filament movement (color-coded) in a notochord cell (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001781#pbio.1001781.s014" target="_blank">Movie S3</a>). The average velocity is 33.9±4.9 nm/s (mean ± s.e.m., <i>n</i> = 9). (F) Kymograph of actin filament movement (indicated by arrows) based on <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001781#pbio.1001781.s014" target="_blank">Movie S3</a> at the location indicated by the dash line in (E). The velocity is 29.5±10.5 nm/s, <i>n</i> = 2. (G and H) Dynamics of actin filament movement revealed by FRAP. (G) A projection of a mCherry-hActin–expressing cell that was photobleached. The photobleach mark was made over the entire equatorial region, and the halftime for recovery was determined in both the whole region, and separately in the anterior (ant), middle (mid), and posterior (post) regions, and is shown in (H). Recovery is faster in the anterior and posterior regions than in the middle region. This confirms that actin filaments flow toward the equator. Actin movement velocity is 40.8±3.8 nm/s, <i>n</i> = 4, calculated from the recovery kinetics of the whole region. Scale bars, 5 µm.</p

Conservation of equatorial actin ring and circumferential contraction as a common and mechanistically scalable biophysical solution for anisotropic shape change.

<p>(A) Confocal sections of elongating notochord cells in an appendicularian <i>Oikopleura dioica</i> embryo stained with phallacidin show the presence of an equatorial constriction (red arrows) and actin ring (white arrows). Yellow arrowhead indicates the actin filaments in muscle sarcomeres. (B) The equatorial circumferential constriction (red arrowhead) created by an actomyosin ring (green lines) in a dividing cell causes cell elongation (brown arrows) before cell division. Blue arrowheads indicate filament flow. (C) In notochord cells, an actomyosin ring (green lines) causes an equatorial constriction (red arrowhead), which contributes to the cell elongation (brown arrows). (D) During <i>C. elegans</i> embryogenesis, numerous circumferential actin filaments (green lines) are present at the outer surface of the hypodermal cells. The elongation of the embryo (brown arrows) is accomplished by the contractility of these actin filaments squeezing the embryo circumferentially (red arrowheads) at the scale of the entire embryo. Scale bar, 5 µm.</p

Localization of actin and actin-binding proteins in the equatorial region of notochord cells.

<p>(A) A diagram of a notochord cell at 19 hpf. The notochord cells have a cylindrical shape, with two lateral domains at the anterior and posterior ends of the cell (designated as poles, arrowheads) to form contact with two adjacent notochord cells, and a single, circumferential, basal domain that contacts the notochordal sheath. During lumen formation the center of lateral domains differentiates into apical domain that contacts emerging lumen. The morphological constriction is located at the equator of the cell (arrow). (B and B′, section and projection) Colocalization of F-actin (green) and pS19 MRLC (red) in the equatorial region of the basal domain (yellow arrows). F-actin and pS19 MRLC are also localized in the lateral domains, where they partially overlap (arrowheads). (C) Maximal projection of notochord cells shows the localization of cofilin, α-actinin, tropomyosin, and talinA revealed by immunohistochemistry. Yellow arrows indicate the accumulation of these proteins in the equatorial region. (D) Maximal projection of notochord cells shows the localization of cofilin-mCherry, α-actinin-mCherry, mCherry-tropomyosin, and mCherry-talinA in live embryos. Whereas mCherry-α-actinin, mCherry-tropomyosin, and mCherry-talinA I/LWEQ localize in a broad region (yellow arrows), cofilin-mCherry consistently localizes in a narrow line at the equator (yellow arrowhead). Anterior to the left. Scale bars, 5 µm.</p

Basal local contractions in notochord cells during elongation.

<p>(A) Time-lapse frames of a lifeact-mEGFP–expressing notochord cell forming a bleb (indicated by an arrowhead) at 17 hpf (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001781#pbio.1001781.s012" target="_blank">Movie S1</a>). Yellow arrow indicates equatorial constriction. (B) Kinetics of the membrane deformation (in % of the maximal deformation) and relative cortical actin intensity (in % of maximal fluorescent intensity) at the bleb. The bleb goes through a fast expansion phase in approximately 27 s (<i>n</i> = 14) and a slow retraction phase in subsequent 182 s. (C) Colocalization of tropomyosin, cofilin, and MRLC with lifeact-mEGFP at a bleb (arrowheads). (D) Time-lapse Nomarski images of a bleb (enlarged in blue box), and the movement of apical/luminal membrane (enlarged in red box) in a notochord cell (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001781#pbio.1001781.s013" target="_blank">Movie S2</a>). (E) The kinetics of basal membrane deformation (blue double-headed arrow in D, expressed as % of the maximal deformation) and luminal membrane displacement (red doubled-headed arrow in D, between apex of luminal domain and artificial line running through the lateral domain, expressed as % of maximal displacement). Temporal cross-correlation analysis of the membrane movements shows a high correlation between temporal profiles of basal blebbing and apical membrane displacement (mean  = 0.76±0.05, <i>n</i> = 4), indicating that they have similar overall dynamics. The bleb retraction precedes the outbound movement of apical membrane by 49.87±23.92 s. (F) A model in which blebbing at the basal circumference causes the apical/luminal membrane to displace along the longitudinal axis. (G) A model in which the local contractile forces from bleb retraction at the basal domain are partially registered by equatorial contractile ring, which serves as a ratchet and contributes to cell elongation. Scale bars, 5 µm.</p

Role of α-actinin in notochord cell elongation.

<p>(A) Structure of <i>Ciona</i> α-actinin and design of the α-actininROD mutant. <i>Ciona</i> α-actinin has two calponin homology domains (CH) at the carboxyl terminus, four spectrin-repeats (Spectrin) in the central region, and calmodulin-like (CaM) domain with two EF-hand motifs at the amino terminus. α-actininROD mutant lacks CH domains that have been shown to mediate actin binding. (B) Phenotype of α-actininROD–expressing notochord cells (maximal projection) at 18 hpf. Cells expressing the α-actininROD mutant have a wedged shape. Circumferential actin filaments are not restricted to the equatorial region. (C) A model of α-actinin (red) regulating the placement of actin filaments (green, showing cross-section) and cell elongation. In normal cells, α-actinin tethers actin filaments to the equatorial membrane through its actin- and membrane-binding activity. α-actininROD mutant fails to associate with actin filaments but nevertheless can occupy the equatorial membrane tether sites, therefore disrupting the anchoring of the actin filaments in the equatorial region, consequently resulting in mislocalized contraction. Scale bar, 5 µm.</p