CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2''s association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.     

c-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells

During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3β1, αVβ3, and αVβ5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3β1, αVβ3, and x-CT receptors but not αVβ5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3β1 and αVβ3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MβCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3β1, -αVβ3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.     

Reactive Oxygen Species Are Induced by Kaposi's Sarcoma-Associated Herpesvirus Early during Primary Infection of Endothelial Cells To Promote Virus Entry

The entry of Kaposi''s sarcoma-associated herpesvirus (KSHV) into human dermal microvascular endothelial cells (HMVEC-d), natural in vivo target cells, via macropinocytosis is initiated through a multistep process involving the binding of KSHV envelope glycoproteins with cell surface α3β1, αVβ3, and αVβ5 integrin molecules and tyrosine kinase ephrin-A2 receptor, followed by the activation of preexisting integrin-associated signaling molecules such as focal adhesion kinase (FAK), Src, c-Cbl, phosphoinositide 3-kinase (PI-3K), and Rho-GTPases. Many viruses, including KSHV, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells; however, the role of ROS in early events of viral entry and the induction of signaling has not been elucidated. Here we show that KSHV induced ROS production very early during the infection of HMVEC-d cells and that ROS production was sustained over the observation period (24 h postinfection). ROS induction was dependent on the binding of KSHV to the target cells, since pretreatment of the virus with heparin abolished ROS induction. Pretreatment of HMVEC-d cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited KSHV entry, and consequently gene expression, without affecting virus binding. In contrast, H₂O₂ treatment increased the levels of KSHV entry and infection. In addition, NAC inhibited KSHV infection-induced translocation of αVβ3 integrin into lipid rafts, actin-dependent membrane perturbations, such as blebs, observed during macropinocytosis, and activation of the signal molecules ephrin-A2 receptor, FAK, Src, and Rac1. In contrast, H₂O₂ treatment increased the activation of ephrin-A2, FAK, Src, and Rac1. These studies demonstrate that KSHV infection induces ROS very early during infection to amplify the signaling pathways necessary for its efficient entry into HMVEC-d cells via macropinocytosis.     

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore‐forming toxins

During infection, plasma membrane (PM) blebs protect host cells against bacterial pore‐forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT‐induced blebbing. Gp96 interacts with non‐muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA–Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod‐like structures from polarized migrating cells, a process that also promotes NMHCIIA–Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO‐dependent killing.     

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Kaposi''s sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2''s tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.     

The Ubiquitin/Proteasome System Mediates Entry and Endosomal Trafficking of Kaposi's Sarcoma-Associated Herpesvirus in Endothelial Cells

Ubiquitination, a post-translational modification, mediates diverse cellular functions including endocytic transport of molecules. Kaposi''s sarcoma-associated herpesvirus (KSHV), an enveloped herpesvirus, enters endothelial cells primarily through clathrin-mediated endocytosis. Whether ubiquitination and proteasome activity regulates KSHV entry and endocytosis remains unknown. We showed that inhibition of proteasome activity reduced KSHV entry into endothelial cells and intracellular trafficking to nuclei, thus preventing KSHV infection of the cells. Three-dimensional (3-D) analyses revealed accumulation of KSHV particles in a cytoplasmic compartment identified as EEA1+ endosomal vesicles upon proteasome inhibition. KSHV particles are colocalized with ubiquitin-binding proteins epsin and eps15. Furthermore, ubiquitination mediates internalization of both KSHV and one of its receptors integrin β1. KSHV particles are colocalized with activated forms of the E3 ligase c-Cbl. Knock-down of c-Cbl or inhibition of its phosphorylation reduced viral entry and intracellular trafficking, resulting in decreased KSHV infectivity. These results demonstrate that ubiquitination mediates internalization of both KSHV and one of its cognate receptors integrin β1, and identify c-Cbl as a potential E3 ligase that facilitates this process.     

Regulation of plasma membrane blebbing by the cytoskeleton

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events.     

Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.     

Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.     

Cell shape and organelle modification in apoptotic U937 cells

U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode) and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis) were simultaneously analyzed.     

EhRho1 regulates plasma membrane blebbing through PI3 kinase in Entamoeba histolytica

The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis. Blebbing is an essential process contributing to cellular motility in many systems. In mammalian cells, formation of plasma membrane blebs is regulated by Rho‐GTPases through its effectors, such as Rho kinase, mDia1, and acto‐myosin proteins. In this study, we have illuminated the role of EhRho1 in bleb formation and motility of E. histolytica. EhRho1 was found at the site of bleb formation in plasma membrane of trophozoites. Overexpression of mutant EhRho1 defective for Guanosine triphosphate (GTP)‐binding or down‐regulating EhRho1 by antisense RNA resulted in reduced blebbing and motility. Moreover, serum‐starvation reduced blebbing that was restored on serum‐replenishment. Lysophosphatidic acid treatment induced bleb formation, whereas wortmannin inhibited the process. In all these cases, concentration of GTP‐EhRho1 (active) and Phosphatidylinositol 4,5‐bisphosphate (PIP2) inversely correlated with the level of plasma membrane blebbing. Our study suggests the role of EhRho1 in blebbing and bleb‐based motility through PI3 kinase pathway in E. histolytica.     

Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Interaction and fusion dynamics between cellular blebs

Membrane blebbing, as a mechanism for cells to regulate their internal pressure and membrane tension, is believed to play important roles in processes such as cell migration, spreading and apoptosis. However, the fundamental question of how different blebs interact with each other during their life cycles remains largely unclear. Here, we report a combined theoretical and experimental investigation to examine how the growth and retraction of a cellular bleb are influenced by neighboring blebs as well as the fusion dynamics between them. Specifically, a boundary integral model was developed to describe the shape evolution of cell membrane during the blebbing/retracting process. We showed that a drop in the intracellular pressure will be induced by the formation of a bleb whose retraction then restores the pressure level. Consequently, the volume that a second bleb can reach was predicted to heavily depend on its initial weakened size and the time lag with respect to the first bleb, all in quantitative agreement with our experimental observations. In addition, it was found that as the strength of membrane-cortex adhesion increases, the possible coalescence of two neighboring blebs changes from smooth fusion to abrupt coalescence and eventually to no fusion at all. Phase diagrams summarizing the dependence of such transition on key physical factors, such as the intracellular pressure and bleb separation, were also obtained.     

Cell Blebbing upon Addition of Cryoprotectants: A Self-Protection Mechanism

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.     

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.     

Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein

Nucleocytoplasmic transport of signaling modulators is essential for regulating cellular responses to extracellular stimulation and stress, as well as pathogen infection. Exportin 1 (XPO1), also known as chromosomal maintenance 1 (CRM1), mediates nuclear export of proteins, rRNAs, snRNAs, and some mRNAs. In this study, we have identified an essential role of XPO1 in regulating Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication during primary infection of primary human umbilical vein endothelial cells. Treatment with an XPO1 inhibitor KPT-8602 and short hairpin RNA (shRNA)-mediated knockdown of XPO1 reduced KSHV lytic replication but had no effect on KSHV entry and trafficking. XPO1 inhibition induced retention of autophagy adaptor protein p62 (SQSTM1) in the nucleus, which enhanced activation of TBK1 and IRF3. As a result, nuclear accumulation of p62 increased expression of innate immune-related genes including IRF7, ISG15, IFIT1, IFIT2, and IFIT3, leading to a reduction of KSHV lytic replication. These results illustrate a novel mechanism by which XPO1 mediates innate immune response and KSHV replication, and identify XPO1 as a potential therapeutic target and KPT-8602 as a promising therapeutic agent for KSHV infection.Subject terms: Mechanisms of disease, Infection     

Differences in cortical actin structure and dynamics document that different types of blebs are formed by distinct mechanisms

Bleb formation has been studied by specifically targeting major factors controlling this process, such as microtubule disassembly, local actin depolymerization, and increased pressure. At least two different types of blebs (types 1 and 2) formed by different mechanisms and possibly a third type (type 3) can be documented at the front of living polarized cells expressing green fluorescent protein-actin and/or in fixed and stained cells. Type 1 blebs (membrane/cortex dissociation blebs) formed by dissociation of the plasma membrane from cortical actin develop cytoplasmic actin layers associated with restriction rings. They can be induced by the microtubule-disassembling agent colchicine. Type 2 blebs (cortical actin disassembly blebs) form after disassembly of the cortical actin layer in the presence of latrunculin A. Restriction rings without a cytoplasmic actin layer occur in a transition zone between the intact cortical actin layer of the cell body and the compromised actin layer of the bleb. Evidence for a third type of bleb (type 3), showing an intact cortical actin layer but no cytoplasmic actin layer and no recognizable relationship between the actin cytoskeleton and the restriction ring, has been obtained by passive cell deformation in micropipettes, which increases pressure. Repolymerization of the cortical actin layer does not necessarily result in bleb retraction. Once formed, restriction rings do not narrow, suggesting that they result from isometric contraction. A simplified classification scheme has been developed to relate the type of bleb to specific signals or cell functions. Its application shows that spontaneously blebbing cells form almost exclusively type 1 blebs.     

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2–Bub2–Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.