Constitutively active Dia alters the F-actin structure at the fusion site and regulates localization of Arp2/3 regulators.

<p><b>A-B.</b> Stage 14 embryos stained for F-actin (phalloidin, white) and for Dia::GFP or DiaΔDAD::GFP (GFP antibody, white); FCM (magenta), FC/Myotube (turquoise). Scale bar: 5μM. <b>A.</b> Control <i>DMef2-Gal4>UAS-dia</i>::<i>GFP</i> myoblasts show colocalization of Dia::GFP and the F-actin focus at the fusion site. <b>B.</b><i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> myoblasts show that F-actin does not form a well-defined focus at the fusion site but appears diffuse. DiaΔDAD::GFP localizes to the plasma membrane and is enriched at cell contact sites. <b>C.</b> Fluorescent intensity curves confirm the distribution of F-actin in embryos expressing Dia::GFP and DiaΔDAD::GFP. <b>D-E.</b> Stage 15 embryos stained for F-actin (phalloidin) and DiaΔDAD::HA (antibodies against HA) showing FCM (magenta) and FC/Myotube (turquoise). Scale bar: 5μM. <b>D.</b><i>DMef2-Gal4</i> driven expression of DiaΔDAD::HA in <i>kette</i>^<i>J4-48</i> mutant background. The morphology of the F-actin focus at the fusion site appears similar to <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryos. While F-actin localizes at the cell cortex, it spreads out at the fusion site and does not make a concentrated focus. <b>E.</b><i>DMef2-Gal4</i> driven expression of DiaΔDAD::GFP in <i>sltr</i>^<i>s1946</i> mutant background. Similar to expression of DiaCA alone, the F-actin localizes at the cell cortex and spreads out at the fusion site. <b>F-G.</b> Stage 14 embryos stained for F-actin (phalloidin), Dia<i>ΔDAD</i>::GFP (GFP antibody) and SCAR or WASp. Scale bar: 5μM. <b>F.</b> SCAR localization in control and <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryos. In control embryos, SCAR accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, SCAR loses its characteristic concentration at the fusion site and becomes found throughout the cytoplasm. The multiple peaks in the SCAR fluorescent intensity curve confirm SCAR’s change in localization. <b>G.</b> Localization of WASp in control and <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryo. In control embryos, WASp accumulates at the fusion site, as confirmed by the fluorescent intensity curves. When expressing DiaΔDAD::GFP, WASp displays a more diffused localization. Fluorescent intensity curves confirm the broader distribution of WASp signal in relation to the controls.</p

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

<div><p>The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for <i>Drosophila</i> myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.</p></div

Dia regulates actin and Arp2/3 activity during myoblast fusion.

<p><b>A.</b> In stage 15 embryos expressing 2x<i>UAS-diaDN</i>::<i>GFP</i>, myoblasts are stained with phalloidin and antibodies against DiaDN::GFP. Immunostaining for Sns and Duf was used to examine cell adhesion. Sns and Duf localize correctly at the fusion site, confirming adhesion between FCMs and FC/myotube. <b>B.</b> Stage 15 embryos expressing 2x<i>UAS-diaDN</i>::<i>GFP</i> with 2x<i>DMef2-Gal4</i>. Myoblasts are stained with phalloidin and antibodies against GFP. Arrowhead points to a myoblast adhering to the myotube but failing to generate a F-actin focus. <b>C.</b> Percentage of fusion sites with and without actin focus. Embryos expressing 2x <i>UAS-diaDN</i>::<i>GFP</i> were stained with phalloidin, and the formation of actin focus was quantified in these embryos. Actin focus forms in 60% of fusion sites and is absent in 40%. <b>D</b>. Time-lapse imaging of DiaDN fusion block. Three copies of <i>UAS-diaDN</i>::<i>GFP</i> and one copy of <i>UAS-moesin</i>::<i>mCherry</i> were driven by two copies of <i>Dmef2-Gal4</i>. F-actin dynamics were visualized by moesin::mCherry. Still images from the time-lapse sequence show 2–3 myoblasts adhered to a myotube (dashed lines), but unable to fuse. No F-actin focus was detected at the fusion site (arrow) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s004" target="_blank">S1 Movie</a>; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s009" target="_blank">S3I Fig</a>). <b>E-F.</b> Comparison of SCAR and WASp localization in DiaDN-expressing FCMs. In embryos expressing high levels of DiaDN, immunostaining was used to examine the localization of SCAR and WASp. At a fusion site in which an actin focus forms (upper panels), SCAR and WASp both correctly localize to the actin focus (arrow; phalloidin). When actin focus formation is disrupted by DiaDN (lower panels), SCAR and WASp no longer accumulate at the fusion site (asterisk). <b>G.</b> Dia localization in <i>sltr</i>^<i>s1946</i><i>; kette</i>^<i>J4-48</i> double mutant. In this double mutant, phalloidin was used to label F-actin, and Dia antibody to detect the localization of Dia. Fusion is blocked in double mutants, with no actin focus forming. Dia still accumulates at the fusion site. <b>H.</b> Dia localization in myoblasts expressing <i>UAS-PH</i>^<i>plcγ</i>::<i>GFP</i>. Stage 15 embryos expressing 2x<i>PH</i>^<i>plcγ</i>::<i>GFP</i> with 2x<i>DMef2-Gal4</i>. Myoblasts are labeled with antibodies against GFP. <i>PH</i>^<i>plcγ</i> sequesters PI(4,5)P2, generating a small actin focus and blocking myoblast fusion<b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.ref054" target="_blank">54</a>]</b>. In an FCM that adheres to a FC, immunostaining reveals that Dia accumulates at the fusion site. Scale bar: 2.5μM.</p

Constitutively active Dia blocks myoblast fusion.

<p><b>A.</b> Schematic diagram of Dia domain structure and different constitutively active Dia deletion constructs used in this study. <b>B.</b> Whole mount lateral view of three hemisegments from stage 16 embryos showing the (MHC) labeled muscles and nuclei (apME-NLS::dsRed) of the lateral transverse (LT) muscles. Scale bar: 24μM. Expression of DiaCA blocks myoblast fusion as visualized by many free myoblasts (arrows). This fusion defect is not due to a failure in FC specification as witnessed by expression of apME-NLS::dsRed in nuclei. <b>C.</b> Fusion index confirms a total block in myoblast fusion: dsRed positive nuclei in LT muscles/ hemisegment were counted in control (26.6±1.5) and <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> (5.2±1.0) (n = 40 hemisegments/genotype) (p<0.001). <b>D.</b> Dynamics of DiaΔDAD::GFP expression in myoblasts. Still images from time-lapse of a stage 14 <i>DMef2-Gal4>UAS-diaΔDAD</i>::<i>GFP</i> embryo. Saturated image shows outline of cells and is used to localize myoblasts attempting to fuse. Filopodia-like protrusions undergo highly dynamic extension and retraction at areas of cell contact. DiaΔDAD::GFP localizes at the tip of those protrusions (arrows), and this signal moves as the filopodium extends and retracts (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s006" target="_blank">S3 Movie</a>). Scale bar: 5μM <b>E.</b> Still images from time-lapse of stage 14 <i>twist-actin</i>::<i>GFP; DMef2-Gal4>UAS-diaFH1FH2</i> embryo. Images at 0 and 3 min show filopodia-like protrusions (arrows) emanating from the FCM, which adheres to the FC but is unable to fuse. The Actin::GFP signal is enriched at the fusion site during the entire time lapse sequence (1 hr). Compare to control FCM (<i>twist-actin</i>::<i>GFP</i>, lower panel), which fuses with FC in 30 minutes. Scale bar: 4μM</p

Dia localization at the fusion site is dependent on FC/FCM recognition and adhesion, but independent of regulators of Arp2/3.

<p>Stage 15 embryos stained with phalloidin (<b>i.</b>), antibodies against Dia (<b>ii.</b>), and Myosin Heavy Chain (<b>iii.</b>, MHC). Phalloidin labels F-actin (focus and sheath) at the fusion site; MHC identifies myoblasts. FCM (magenta) and FC/Myotube (turquoise). <b>A-iv.</b> Dia localization in FCM and FC/myotube in a wild-type embryo during myoblast fusion. Dia accumulates at the fusion site. The averaged fluorescence intensity curve (Aiv, n = 5) in wild-type embryos confirms Dia colocalization with actin. <b>B-iv.</b> In <i>sns</i> mutants, no F-actin focus is formed and no specific accumulation of actin or Dia are observed. Average fluorescence intensity curve of <i>sns</i> mutant embryos (Biv, n = 5) supports that Dia does not accumulate at the fusion site and is cytoplasmic. <b>C-E-iv.</b> In <i>rac</i>, <i>mbc</i>, and <i>kette</i> mutants, SCAR activity is lost, an enlarged focus is observed at the fusion site, and Dia is enriched at the fusion site. Fluorescence intensity curves confirm Dia and actin colocalization in <i>rac</i> (Civ), <i>mbc</i> (Div), and <i>kette</i> (Eiv) mutants (n = 5/genotype). <b>F-iv.</b> In <i>loner</i> mutant embryos, Dia accumulates at the F-actin focus, as confirmed by the fluorescence intensity curves (n = 5). <b>G-I-iv.</b> In <i>blow</i>, <i>sltr(Dwip)</i> and <i>wsp</i> mutants, where WASp-mediated actin remodeling is lost, Dia accumulation at the fusion site is unaffected. Fluorescence intensity curves confirm the colocalization of Dia and F-actin in <i>blow</i> (Giv), <i>sltr(Dwip)</i> (Hiv), <i>and wsp</i> (Iiv) mutants (n = 5/genotype). Scale bar: 2.5μM.</p

Model: Dia and Arp2/3 function together to regulate myoblast fusion.

<p>During myoblast fusion, the transmembrane molecules (e.g. Sns and Duf) mediate recognition and adhesion between the FCM and the FC/myotube. After cell adhesion, Dia is recruited to the fusion site, where it collaborates with PI(4,5)P2 signaling in making a functional F-actin focus, How PI(4,5)P2 signaling coordinates with Dia is unclear, but it may possibly be through the recruitment of small GTPases that activate Dia. Active Dia, in turn, nucleates F-actin particularly on the FCM side, which serves as a substrate for Arp2/3. Coordinated Dia and Arp2/3 activities allow the actin network to consolidate into invasive podosome-like structures. Loss of filamentous actin and reduction in Arp2/3 NPFs recruitment/maintenance due to Dia loss results in Arp2/3 being unable to nucleate enough actin filaments to build an invasive podosome-like structure. Hence no fusion can occur. In Dia gain of function embryos, Dia builds excessive actin filaments. Arp2/3 NPFs fail to localize properly, leading to an alteration in the distribution of activated Arp2/3. Since the actin network fails to consolidate into invasive podosome-like structures, no myoblast fusion can occur.</p

Diaphanous is required for myoblast fusion.

<p><b>A.</b> Schematic diagram of Dia domain structure and a deletion construct that renders Dia dominant negative (DiaDN). DiaDN consists of the FH1 domain and a partially deleted FH2 domain; the deleted aa 750–770 in the FH2 domain is indicated by the dashed area. <b>B</b>. Expression of DiaDN reduces filopodia number in S2R+ cells. S2R+ cells that were transfected with DiaDN::GFP (green in merge, grey in single channel) have less filopodia-like, protrusive structures (phalloidin, red in merge, grey in single channel) relative to untransfected cells (n = 20, p<0.001). Scale bar: 10μM. This reduction was rescued by expression of Dia::GFP (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s009" target="_blank">S3C Fig</a>). <b>C-Ci.</b><i>UAS-diaDN</i>::<i>GFP</i> was expressed in leading edge cells using <i>wg-Gal4</i>. Moesin::mCherry was also expressed in leading edge cells to visualize actin. In stage 15, GFP-negative control cells, filopodia structures are seen (C, arrow). DiaDN::GFP significantly reduced filopodia formation (Ci). Scale bar: 2.5μM. <b>Cii.</b> Filopodium number was quantified in each wg-Gal4 expressing stripe. DiaDN significantly reduced filopodia formation in leading edge cells relative to control (2.1±1.29μM vs 3.95±1.15μM, p<0.001). <b>D.</b> Increasing DiaDN concentration in myoblasts through higher temperature and genetic copy number leads to an increased fusion block. Three hemisegments of a lateral view of a stage 16 embryos stained with GFP and MHC antibody are shown. Myoblast fusion is relatively normal in 1x<i>DMef2-Gal4></i>1x<i>diaDN</i>::<i>GFP</i> embryos at 29°C (upper panel), with few free myoblasts (arrow). In 2x<i>DMef2-Gal4></i>2x<i>diaDN</i>::<i>GFP</i> embryos (lower panel), a higher degree of fusion block (arrows) and muscle detachment (arrowheads) are observed. <b>E.</b> One hemisegment of stage 17 embryo showing apME-NLS::dsRed labeled nuclei in the four lateral transverse (LT) muscles: From left to right: apME-NLS::dsRed labeled nuclei in stage 17 LT muscles in control, in <i>1xDMef2-Gal4> 1xUAS-diaDN</i>::<i>GFP</i>, and in 2x<i>DMef2-Gal4> 2xUAS-diaDN</i>::<i>GFP</i> embryos <b>F.</b> Fusion index of Stage 17 lateral transverse (LT) muscles confirms the degree of fusion block in DiaDN embryos. In control embryos, 27.1±2.3 nuclei were counted in each hemisegment (n = 40 hemisegments). 1x<i>DMef2-Gal4></i> 1x<i>UAS-diaDN</i>::<i>GFP</i> reduces the number of dsRed positive nuclei in each hemisegment to 22.5±2.2 (p<0.001), whereas 2x<i>DMef2-Gal4></i> 2x<i>UAS-diaDN</i>::<i>GFP</i> further reduces the number to 15.5±2.7(p<0.001). Scale bar: 24μM.</p

Diaphanous is localized to the fusion site.

<p><b>A-D.</b> Dia colocalizes with the actin focus at the site of fusion. <b>A.</b> Fusing myoblast (FCM, false colored magenta in all Figures) and FC/Myotube (false colored turquoise in all Figures, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#sec014" target="_blank">Methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s007" target="_blank">S1 Fig</a>) in a stage 15 <i>twist-actin</i>::<i>GFP</i> embryo stained for F-actin (phalloidin, white) and antibodies against Dia (red) and GFP (green). Dia accumulates at the fusion site, colocalizing with the F-actin focus. <b>B.</b> Signal intensity plot confirms Dia enrichment with actin at the fusion site. Average fluorescence intensity measured across the F-actin foci as shown in A. A line of predetermined length was dropped across the fusion site; fluorescence intensity along the line was measured in different channels, normalized, and plotted (n = 10). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#sec014" target="_blank">Methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005381#pgen.1005381.s008" target="_blank">S2 Fig</a> for details on intensity measurements and normalization. <b>C.</b> Fluorescent intensity curves with error bars for both control (n = 10) and proteins of interest (n = 10). <b>D.</b> Still images from a time-lapse series of a fusion event in a stage 14 embryo expressing Dia::GFP and Moesin::mCherry driven by <i>DMef2-Gal4</i> indicates that Dia::GFP has the same spatial and temporal pattern as actin at the fusion site. Moesin::mCherry (red) labels F-actin at the fusion site (arrows). <b>Di.</b> Signal intensity curve showing Dia::GFP (green) and Moesin::mCherry (red) colocalize during fusion at each time point. <b>E-H.</b> Dia is localized to the fusion site in both the FC/Myotube (turquoise) and FCM (magenta). Dia::GFP (green), phalloidin (white), endogenous Dia (red) <b>E.</b><i>duf5</i>.<i>1-Gal4</i> driven Dia::GFP shows expression in myotubes/FCs in stage 14 embryos. Fusing FCM and myotube were captured before cytoplasmic mixing. Dia antibody staining (red) is present at the F-actin focus (phalloidin, white). FC driven Dia::GFP (green) expressed in FC/Myotubes partially overlaps with endogenous Dia at the fusion site. <b>F.</b> The signal intensity curve confirms that the peak of FC driven Dia::GFP partially overlaps with endogenous Dia. <b>G.</b> Stage 16 <i>sns-Gal4</i> driven Dia::GFP shows expression in <i>mbc</i>^<i>C1</i> mutant FCMs, where fusion is blocked. Staining as in F. Dia enrichment is seen on FCM side with both Dia (red) and FCM driven Dia::GFP (green). <b>H.</b> Signal intensity curve confirms Dia::GFP and Dia overlap and are within the F-actin peak. Scale bar: 2.5μM</p

Constitutively active Diaphanous accelerates the actin exchange rate at the fusion site.

<p>Fluorescence recovery of Actin::GFP after photobleaching. Newly formed Actin::GFP foci in stage 14 embryos were photobleached (arrows) to approximately 30% of the original intensity. The recovery rate was recorded every 3s after photobleaching for a total of 165 sec (endpoint). <b>A.</b> Stills from time-lapse showing Actin::GFP recovery at an actin focus after photobleaching in wild-type and <i>DMef2-Gal4> UAS-diaFH1FH2</i> embryos. Scale bar: 2.5μM <b>B.</b> Comparison of representative recovery kinetics of Actin::GFP foci in control (green) and <i>DMef2-Gal4> UAS-diaFH1FH2</i> (red) myoblasts. <b>C.</b> Half time of fluorescent recovery in control (n = 8) and <i>DMef2-Gal4>UAS-diaFH1FH2</i> embryos (n = 10). The half time of fluorescence recovery in <i>DMef2-Gal4> UAS-diaFH1FH2</i> embryos (t_1/2 = 17.7±6.7s) is significantly lower than control (t_1/2 = 53.3±6.7s). <b>D.</b> Percentage of final recovery in control and <i>DMef2-Gal4>UAS-diaFH1FH2</i> embryos. Final recovery in <i>DMef2-Gal4> UAS-diaFH1FH2</i> embryos (77.9±10.6%) is similar to wild-type embryos (78.6±10.5%) (p>0.1).</p

A Geometric Representation of Collective Attention Flows

<div><p>With the fast development of Internet and WWW, “information overload” has become an overwhelming problem, and collective attention of users will play a more important role nowadays. As a result, knowing how collective attention distributes and flows among different websites is the first step to understand the underlying dynamics of attention on WWW. In this paper, we propose a method to embed a large number of web sites into a high dimensional Euclidean space according to the novel concept of flow distance, which both considers connection topology between sites and collective click behaviors of users. With this geometric representation, we visualize the attention flow in the data set of Indiana university clickstream over one day. It turns out that all the websites can be embedded into a 20 dimensional ball, in which, close sites are always visited by users sequentially. The distributions of websites, attention flows, and dissipations can be divided into three spherical crowns (core, interim, and periphery). 20% popular sites (Google.com, Myspace.com, Facebook.com, etc.) attracting 75% attention flows with only 55% dissipations (log off users) locate in the central layer with the radius 4.1. While 60% sites attracting only about 22% traffics with almost 38% dissipations locate in the middle area with radius between 4.1 and 6.3. Other 20% sites are far from the central area. All the cumulative distributions of variables can be well fitted by “S”-shaped curves. And the patterns are stable across different periods. Thus, the overall distribution and the dynamics of collective attention on websites can be well exhibited by this geometric representation.</p></div