Upstream activators of Rho GTPase (MK2 EE and hsp27 DDD) disrupt p-bodies.

<p>A–D) HUVECs were transduced with recombinant retroviruses that express a constitutively active version of MK2 (MK2 EE, Flag-tagged) or phosphomimicking heat shock protein (hsp)27 (hsp27 DDD, HA-tagged). Following two-day selection with puromycin, cells were seeded onto coverslips. The next day, cells were either not treated or treated with C3 transferase (in basal media) for 6 hours at 37°C to irreversibly inactivate Rho GTPase. After the 6-hour incubation, cells received one hour of normal media before fixation, permeabilization, and staining with the following primary antibodies: mouse anti-hedls or rabbit anti-DDX6 (p-bodies, green), rabbit anti-HA (hsp27 DDD-HA, false-colored blue), or mouse anti-Flag (MK2 EE-Flag, false-colored blue). To visualize actin stress fibers, cells were also labeled with phalloidin (red). To quantify p-body disruption, the number of cells expressing MK2 EE or hsp27 DDD that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a>. n = 3 independent experiments Scale bar  = 10 µm. E) HUVECs were sequentially transduced with two populations of recombinant retroviruses: firstly, puromycin-resistant viruses that express MK2 EE-Flag, hsp27 DDD-HA or the empty vector; and secondly, blasticidin-resistant viruses that express a dominant negative version of Rho (Rho DN-HA tagged) or the empty vector. At each step, positive transductants were selected with the appropriate drug for 2 days. Following this, cells were seeded onto coverslips and the next day, treated with basal media for 1 hour before fixation, permeabilization and staining as described. To quantify p-body disruption, the number of cells expressing the transgene of interest that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a> (n = 2 independent experiments).</p

Model describing the effects of KapB in altering endothelial cell cytoskeleton and reprogramming gene expression by disrupting P-bodies and stabilizing ARE-mRNAs.

<p>When the p38/MK2 pathway is not activated by stress or KapB expression (cell on the left), MK2 remains in the nucleus, hsp27 remains bound to actin filaments, p115RhoGEF is found in a cytosolic oligomer-induced inhibitory state, and RhoA is not active. In this case, the actin cytoskeleton is unchanged, p-bodies are not dispersed and ARE-containing RNAs remain unstable. When KapB is expressed (cell on the right), it mimics the stress-induced activation of the p38/MK2 pathway, binds to MK2 and stimulates its kinase activity, resulting in hsp27 phosphorylation and the stimulation of p115RhoGEF activity, leading to RhoA activation. The consequences of the dual stimulation of RhoA and MK2 by KapB are as follows: 1. RhoA and ROCK-dependent formation of actin stress fibers, and migratory and angiogenic phenotype and 2. RhoA-dependent but ROCK-independent dispersal of PBs that correlates with the increased stability of ARE-containing mRNAs and the translation of their encoded proinflammatory and angiogenic protein products.</p

KapB requires the kinase MK2 and RhoA-GTPase to induce actin stress fibers in primary endothelial cells.

<p>A) HUVECs expressing KapB (panels a–d) or controls (MK2-EE [panels e–h] or hsp27-DDD [panels i–l]) were treated with inhibitors to examine the roles of the kinases p38 and MK2 and the GTPase RhoA in stress fiber formation. Cells were either treated for one hour with 3 µM of the p38 kinase inhibitor SB203580, 10 µM of the MK2 inhibitor, Inhibitor III, 10 µM of the rho-associated kinase (ROCK1/2) inhibitor, Y-27632, or DMSO as a vehicle control, before fixation and staining as above. B) To examine activation of the RhoA GTPase by KapB and controls, HeLa-Tet Off cells were transfected with an expression plasmids for KapB, MK2-EE, hsp27-DDD or an empty vector control and assayed for active (GTP-bound) RhoA using the Active Rho Detection Kit (CST) according to manufacturer's instructions. Before lysis, transfected cells were starved in low serum media for a total of 24–28 hours and either treated or not treated with the RhoA activator lysophosphatidic acid (LPA) for 3 minutes. Total cell lysate and pull-downs containing the active (GST-bound) form of RhoA were subjected to SDS-PAGE and immunoblotted with anti-RhoA (CST). One representative experiment of three is shown.</p

KapB-mediated activation of RhoA-GTPase and PB dispersal requires MK2.

<p>HUVECs were sequentially transduced with two populations of recombinant retroviruses: firstly, puromycin-resistant viruses that express either a short hairpin RNAs (shRNAs) against MK2 or the scrambled (scr) shRNA control, and secondly, viruses that express KapB or the empty vector. After the first step, positive transductants were selected with puromycin for 2 days. A) To examine MK2 expression after knock-down, HUVECs were washed with PBS and lysed in 1x SDS-protein sample buffer containing protease inhibitors and processed for SDS-PAGE and immunoblotting using anti-MK2 and anti-beta-actin. One representative blot of two is shown. B) To assess RhoA activity, transduced HUVECs were assayed for active (GTP-bound) RhoA using the Active Rho Detection Kit (CST) according to manufacturer's instructions. Confluent 6-well plates of HUVECs cells were used for the assay. Total cell lysate (1/10) and pull-downs containing the active (GST-bound) form of RhoA were subjected to SDS-PAGE and immunoblotted with anti-RhoA. One representative experiment is shown. C–D) To examine PBs, transduced cells were seeded onto coverslips. KapB-expressing cells were identified by positive immunoflurescent staining with anti-KapB. To quantify p-body disruption, the number of cells expressing KapB that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a> (n = 3 independent experiments; Scale bar  = 10 µm).</p

KapB promotes actin stress fiber formation in human umbilical vein endothelial cells.

<p>Expression of KapB (panel b) or controls (a constitutively active version of MK2 [MK2-EE, panel c], a phosphomimicking version of hsp27 [hsp27-DDD, panel d] or an empty vector control [panel a]) in HUVECs was achieved by retroviral transduction and a 2-day selection with puromycin, after which cells were split onto coverslips and processed for fluorescence microscopy. Cells were fixed in 4% paraformaldehyde (in PBS) permeabilized in 0.1% Triton X-100 (in PBS) and stained with Alexa 555-conjugated to phalloidin to visualize the actin cytoskeleton (panels a-d). MK2-EE and hsp27-DDD have previously been shown to induce actin stress fiber formation in endothelial cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat.1004597-Kayyali1" target="_blank">[59]</a>. Scale bar  = 10 µm.</p

KapB disperses p-bodies.

<p>A–B) HUVECs transduced to express of KapB or vector control were split onto coverslips and processed for immune fluorescence. Cells were fixed 4% paraformaldehyde (in PBS) permeabilized in 0.1% Triton X-100 (in PBS), and blocked in 1% human AB serum before staining with the following primary antibodies: anti-hedls (p-bodies, green) and anti-KapB (KapB, false-coloured blue). To visualize actin stress fibers, cells were also labeled with Alexa 647-conjugated phalloidin (red). To quantify the effect on KapB expression on p-bodies, the number of KapB-expressing cells that displayed p-bodies of normal size (>300 nm in diameter) was counted and compared to control cells. Five independent experiments were performed. >100 cells were counted in each experiment. Scale bars  = 10 µm. C) To stain the microtubule cytoskeleton, cell fixation was performed in pre-warmed, 37°C 4% paraformaldehyde (in D-PBS) for 10 minutes, before permeabilization and immunostaining using anti-KapB (false-colored blue) or anti-tubulin (microtubules, green). Scale bars  = 10 µm.</p

KapB-mediated activation of RhoA-GTPase is important for stabilization of ARE-containing mRNA.

<p>A) HeLa Tet-Off cells were co-transfected with pTRE2-Rluc (no ARE), pTRE2-Fluc-ARE, and an expression plasmids for KapB, the constitutively active forms of RhoA (Rho CA) and MK2 (MK2-EE), the phosphomimicking form of hsp27 (hsp27-DDD) or an empty vector control. At 24 hours post transfection, Dox was added to halt reporter gene transcription. 24 hours after Dox addition, cell lysates were harvested and normalized (firefly/renilla activity) luciferase activity was calculated. Results are displayed in relative light units (RLUs) and are the average of three independent experiments +/− the standard error. B) HeLa tet-off cells were co-transfected with pTRE2-Rluc (no ARE), pTRE2-Fluc-ARE, and an expression plasmids for KapB, the constitutively active form of MK2 (MK2-EE), the phosphomimicking form of hsp27 (hsp27-DDD) or an empty vector control. Transfections were performed with or without the addition of an expression plasmid for the dominant negative form of RhoA (Rho DN). At 24 hours post transfection, Dox was added to halt reporter gene transcription. 24 hours after Dox addition, cell lysates were harvested and analyzed for normalized (firefly/renilla activity) luciferase activity according to the methods. Results are displayed in relative light units (RLUs) and are the average of three independent experiments +/− the standard error. C) HUVECs were transduced with recombinant retroviruses to express KapB, MK2-EE, or empty vector, and selected with puromycin. 24 hours after selection, transduced cells were washed with PBS and lysed in 1x SDS-protein sample buffer containing protease inhibitors and processed for SDS-PAGE and immunoblotting using anti-phosphorylated (serine 82) hsp27 and anti-actin. One representative blot of three independent experiments is shown.</p

KSHV-mediated p-body dispersion in latently infected endothelial cells requires kaposin expression.

<p>A, D) HUVEC cells were either not infected or infected with KSHV for 24, 48, or 72 hours before being fixed with 4% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100 (in PBS), and blocked in 1% human AB serum before staining with the following primary antibodies: anti-hedls (p-bodies, green) and anti-LANA (to mark infected cells, red). In part A, infected cells are denoted with a thin arrow; uninfected cells are marked with a thick arrow. In part D, actin stress fibers were also labeled with phalloidin (false-colored white). B–C) To quantify the effect of latent infection on p-bodies, the number of LANA-expressing cells that displayed p-bodies of normal size (>300 nm in diameter) were counted and compared to uninfected cells. Three independent experiments were performed. >100 cells were counted in each experiment. C–D) Before KSHV infection HUVECs were transduced with two different recombinant lentiviruses that express a short hairpin RNAs (shRNAs) against the kaposin transcript (named shKap1 and shKap2) or the non-specific (NS) shRNA control. Positive transductants were selected by puromycin treatment for two days and then seeded on coverslips for infection with KSHV the next day. 48 hours post-infection, cells were fixed and processed for immune fluorescence as described above. n = 3 independent experiments Scale bar  = 10 µm.</p

Knockdown of the Rho guanine exchange factor (GEF) p115 prevents KapB-induced modification of p-body dynamics.

<p>A-B) HUVECs were sequentially transduced with two populations of recombinant viruses: firstly, puromycin-resistant viruses that express KapB or the empty vector; and secondly, GFP-expressing lentiviruses that express short hairpin RNAs (shRNAs) against a two different Rho guanine exchange factors (GEFs; p115 [numbered −3, −4, and −9], H1 [numbered −1, −2, and −7], or the non-specific (NS) shRNA control. Positive transductants were selected by puromycin treatment (1^st step) and positive GFP-expression, to mark shRNA-expressing cells (2^nd step). After seeding cells on coverslips, and a one-hour treatment in basal media the following day, cells were stained with the following primary antibodies: mouse anti-hedls (to stain p-bodies, false-colored blue) and rabbit anti-KapB (to stain KapB, red). Representative IF images are shown in A. To quantify p-body disruption, the number of cells expressing the transgene of interest (red) and the shRNA construct (green) that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a> and shown in B (n = 3 independent experiments). Scale bar  = 10 µm.</p

KapB activation of RhoA-GTPase but not the downstream kinase ROCK is necessary for p-body disruption.

<p>A-D) HUVECs were transduced with recombinant retroviruses to express eGFP, a constitutively active version of Rho (RhoCA-eGFP), a dominant negative version of Rho (RhoDN-eGFP), KapB, or empty vector. Following two-day selection with puromycin, cells were seeded onto coverslips. The next day, cells were either not treated or treated with C3 transferase (in basal media) for 6 hours at 37°C to irreversibly inactivate Rho GTPase. After the 6-hour incubation, cells received one hour of normal media before fixation, permeabilization, and staining as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a>. Alternately, cells were treated with the specific inhibitor of the Rho kinase ROCK 1/2 (10 µM of Y-27632) for one hour at 37°C before immunostaining. To quantify p-body disruption, the number of cells expressing KapB or controls that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a>. n = 3 independent experiments; Scale bar  = 10 µm. E) HUVECs were sequentially transduced with two populations of recombinant retroviruses: firstly, puromycin-resistant viruses that express KapB or the empty vector; and secondly, blasticidin-resistant viruses that express a dominant negative version of Rho (Rho DN-HA tagged) or the empty vector. At each step, positive transductants were selected with the appropriate drug for 2 days. Following this, cells were seeded onto coverslips and the next day, treated with basal media for 1 hour before fixation, permeabilization and staining as described. To quantify p-body disruption, the number of cells expressing the transgene of interest that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a> (n = 2 independent experiments).</p