Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.     

Pseudorabies virus US3 protein kinase mediates actin stress fiber breakdown

Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus, induced actin stress fiber breakdown. In the present study, using several PRV deletion mutants, we found that the US3 serine/threonine (S/T) protein kinase is involved in breakdown of actin stress fibers in different PRV-infected cell lines. Further, by transfection assays, we showed that PRV US3 itself, in the absence of other viral proteins, is able to trigger actin stress fiber breakdown when it is localized in sufficient amounts in the nucleus.     

Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton

Regulation of the actin cytoskeleton by microtubules is mediated by the Rho family GTPases. However, the molecular mechanisms that link microtubule dynamics to Rho GTPases have not, as yet, been identified. Here we show that the Rho guanine nucleotide exchange factor (GEF)-H1 is regulated by an interaction with microtubules. GEF-H1 mutants that are deficient in microtubule binding have higher activity levels than microtubule-bound forms. These mutants also induce Rho-dependent changes in cell morphology and actin organization. Furthermore, drug-induced microtubule depolymerization induces changes in cell morphology and gene expression that are similar to the changes induced by the expression of active forms of GEF-H1. Furthermore, these effects are inhibited by dominant-negative versions of GEF-H1. Thus, GEF-H1 links changes in microtubule integrity to Rho-dependent regulation of the actin cytoskeleton.     

Targeting of the Sendai virus C protein to the plasma membrane via a peptide-only membrane anchor

Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPSFLKKILKLRGRR . . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic alpha-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.     

Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.     

Vascular adhesion protein 1 mediates binding of immunotherapeutic effector cells to tumor endothelium

Tumor-infiltrating lymphocytes (TIL) can be used as an immunotherapeutic tool to treat cancer. Success of this therapy depends on the homing and killing capacity of in vitro-activated and -expanded TIL. Vascular adhesion protein 1 (VAP-1) is an endothelial molecule that mediates binding of lymphocytes to vessels of inflamed tissue. Here, we studied whether VAP-1 is involved in binding of TIL, lymphokine-activated killer (LAK) cells, and NK cells to vasculature of the cancer tissue. We demonstrated that VAP-1 is expressed on the endothelium of cancer vasculature. The intensity and number of positive vessels varied greatly between the individual specimens, but it did not correlate with the histological grade of the cancer. Using an in vitro adhesion assay we showed that VAP-1 mediates adhesion of TIL, LAK, and NK cells to cancer vasculature. Treatment of the tumor sections with anti-VAP-1 Abs diminished the number of adhesive cells by 60%. When binding of different effector cell types was compared, it was evident that different cancer tissues supported the adhesion of TIL to a variable extent and LAK cells were more adhesive than TIL and NK cells to tumor vasculature. These data suggest that VAP-1 is an important interplayer in the antitumor response. Thus, by up-regulating the expression of VAP-1 in tumor vasculature, it can be possible to improve the effectiveness of TIL therapy.     

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.     

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.     

The enteropathogenic E. coli effector EspH promotes actin pedestal formation and elongation via WASP-interacting protein (WIP)

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades.     

Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane

Introduction of activated p21-activated kinase (PAK) is sufficient to release primary endothelial cells from contact inhibition of growth. Confluent cells display deficient activation of PAK and translocation of Rac to the plasma membrane at matrix adhesions. Targeting Rac to the plasma membrane rescues these cells from contact inhibition. PAK's ability to release human umbilical vein endothelial cells from contact inhibition is blocked by an unphosphorylatable form of its target Merlin, suggesting that PAK promotes mitogenesis by phosphorylating, and thus inactivating, Merlin. Merlin mutants, which are presumed to exert a dominant-negative effect, enable recruitment of Rac to matrix adhesions and promote mitogenesis in confluent cells. Small interference RNA-mediated knockdown of Merlin exerts the same effects. Dominant-negative Rac blocks PAK-mediated release from contact inhibition, implying that PAK functions upstream of Rac in this signaling pathway. These results provide a framework for understanding the tumor suppressor function of Merlin and indicate that Merlin mediates contact inhibition of growth by suppressing recruitment of Rac to matrix adhesions.     

SWIP mediates retromer-independent membrane recruitment of the WASH complex

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P₂) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.     

MAC-1 mediates adherence of human monocytes to endothelium via a protein kinase C dependent mechanism

Leukocyte adherence is mediated by a superfamily of glycoproteins denoted LFA-1 (the lymphocyte function-associated antigen-1), Mac-1 (macrophage antigen-1) and p150,95. The relative importance of these in mediating human monocyte adherence to endothelium, and the biochemical mechanisms which modulate these events, are not understood. In this report, the role of protein kinase C (pkC) in regulating human monocyte adherence to endothelial cells has been investigated. Addition of phorbol 12,13-dibutyrate (PDBu), which specifically stimulates pkC, caused a dose-dependent increase in their adherence to monolayers of bovine aortic endothelial cells. 4 alpha-phorbol didecanoate (4 alpha-PDD), a structural analogue of PDBu which does not stimulate pkC, failed to increase monocyte adhesion. PDBu also produced a dose-dependent increase in the expression of both Mac-1 and p150,95. The pkC-stimulated adherence of monocytes to endothelium was inhibited by the presence of a monoclonal antibody to Mac-1, while monoclonal antibodies to p150,95 and LFA-1 did not influence adherence. It is concluded that monocyte adherence to endothelial cells is regulated through a pkC-dependent mechanism; moreover, this process is mediated primarily via the Mac-1 adhesion glycoprotein.     

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F-actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho-kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia-like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.     

ATP-binding cassette transporter isoform C2 localizes to the apical plasma membrane via interactions with scaffolding protein

ATP-binding cassette transporter isoform C2 (ABCC2) localizes to the apical plasma membrane in polarized cells. Apical localization of ABCC2 in hepatocytes plays an important role in biliary excretion of endobiotics and xenobiotics, but the mechanism by which ABCC2 localizes to the apical membrane has not been conclusively elucidated. Here, we investigate the role of scaffolding proteins on ABCC2 localization with a focus on the function of PDZK1 (post-synaptic density 95/disk large/zonula occludens-1 domain containing 1) in regulating ABCC2 localization. The C-terminal 77 residues of ABCC2 were used to probe interacting proteins from HepG2 cells. Protein mass fingerprinting identified PDZK1 as a major interacting protein. PDZK1 associated with the plasma membrane, most likely at the apical vacuoles of HepG2 cells. Affinity pull-down assays confirmed that the C-terminal NSTKF of ABCC2 bound to the fourth PDZ domain of PDZK1. Removal of this PDZ-binding motif significantly reduced the normal apical localization of ABCC2. In HepG2 cells, overexpression of this fourth domain overcame endogenous PDZK1 and reduced the ABCC2 localization at the apical membrane with a reciprocal increase of intracellular accumulation of mislocalized ABCC2. These results suggest a possible role for an interaction between ABCC2 and PDZK1 in apical localization of ABCC2 in hepatocytes.     

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.     

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate−cystine antiporter system x_c⁻. GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca²⁺ channel ORAI1, but not the Ca²⁺ sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca²⁺-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca²⁺ entry through ORAI1 mediates the detrimental Ca²⁺ entry in programmed cell death induced by GSH depletion. As this detrimental Ca²⁺ influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.     

Actin stress fiber retraction and aggresome formation is a common cellular response to actin toxins

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.     

Rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with syntaxin 1a

Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane syntaxin 1a in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and syntaxin 1a. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or syntaxin 1a. Granuphilin directly binds to the H3 domain of syntaxin 1a containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and syntaxin 1a. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.     

Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae

The Niemann-Pick C1 (NPC1) protein regulates cholesterol transport from late endosomes-lysosomes to other intracellular compartments. In this article, cholesterol transport to caveolin-1 and caveolin-2 containing compartments, such as the trans-Golgi network (TGN) and plasma membrane caveolae, was examined in normal (NPC+/+), NPC heterozygous (NPC+/-), and NPC homozygous (NPC-/-) human fibroblasts. The expression and distribution of NPC1 in each cell type were similar, and characterized by a finely dispersed, granular staining pattern. The expression of caveolin-1 and caveolin-2 was increased in NPC+/- and NPC-/- fibroblasts, although the distribution in each cell type was similar and characterized by predominant staining of the TGN and plasma membrane. The TGN in NPC+/+ fibroblasts was relatively cholesterol-enriched, whereas the TGN in NPC+/- and NPC-/- fibroblasts was partially or completely cholesterol-deficient, respectively. Consistent with studies demonstrating the transport of cholesterol from the TGN to plasma membrane caveolae, the concentration of cholesterol in plasma membrane caveolae isolated from NPC+/- and NPC-/- fibroblasts was significantly decreased, even though the total concentration of plasma membrane cholesterol in each cell type was similar.These studies demonstrate that NPC1 regulates cholesterol transport to caveolin-1 and caveolin-2 containing compartments such as the TGN and plasma membrane caveolae.