Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called ‘invasome’. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal ‘Bep intracellular delivery’ (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal ‘filamentation induced by cAMP’ (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.Author SummaryA wide variety of bacterial pathogens evolved numerous virulence factors to subvert cellular processes in support of a successful infection process. Likewise, bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type-IV-secretion system into infected cells in order to interfere with host signaling processes involved in cytoskeletal dynamics, apoptosis control, and innate immune responses. In this study, we demonstrate that BepC triggers actin stress fiber formation and a linked cell fragmentation phenotype resulting from distortion of rear-end retraction during cell migration. The ability of BepC to induce actin stress fiber formation is directly associated with its ability to bind GEF-H1, an activator of the RhoA pathway that is sequestered in an inactive state when bound to microtubules, but becomes activated upon release to the cytoplasm. Our findings suggest that BepC is anchored via its BID domain to the plasma membrane where it recruits GEF-H1 via its FIC domain, eventually activating the RhoA/ROCK signaling pathway and leading to stress fiber formation.

Fasudil Promotes BMSC Migration via Activating the MAPK Signaling Pathway and Application in a Model of Spinal Cord Injury

Bone marrow-derived mesenchymal stem cells (BMSCs) are considered as transplants for the treatment of central nervous system (CNS) trauma, but the therapeutic effect is restricted by their finite mobility and homing capacity. Fasudil (FAS), a potent Rho kinase inhibitor, has been reported to alleviate nerve damage and induce the differentiation of BMSCs into neuron-like cells. However, the effect of FAS on the migration of BMSCs remains largely unknown. The present study revealed that FAS significantly enhanced the migration ability and actin stress fiber formation of BMSCs in vitro with an optimal concentration of 30 μmol/L. Moreover, we found that activation of the MAPK signaling pathway was involved in these FAS-mediated phenomena. In vivo, cells pretreated with FAS showed greater homing capacity from the injection site to the spinal cord injury site. Taken together, the present results indicate that FAS acts as a promoting factor of BMSC migration both in vitro and in vivo, possibly by inducing actin stress fiber formation via the MAPK signaling pathway, suggesting that FAS might possess synergistic effect in stem cell transplantation of CNS trauma.

Icariin Promotes the Migration of BMSCs In Vitro and In Vivo via the MAPK Signaling Pathway

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in tissue engineering for regenerative medicine due to their multipotent differentiation potential. However, their poor migration ability limits repair effects. Icariin (ICA), a major component of the Chinese medical herb Herba Epimedii, has been reported to accelerate the proliferation, osteogenic, and chondrogenic differentiation of BMSCs. However, it remains unknown whether ICA can enhance BMSC migration, and the possible underlying mechanisms need to be elucidated. In this study, we found that ICA significantly increased the migration capacity of BMSCs, with an optimal concentration of 1 μmol/L. Moreover, we found that ICA stimulated actin stress fiber formation in BMSCs. Our work revealed that activation of the MAPK signaling pathway was required for ICA-induced migration and actin stress fiber formation. In vivo, ICA promoted the recruitment of BMSCs to the cartilage defect region. Taken together, these results show that ICA promotes BMSC migration in vivo and in vitro by inducing actin stress fiber formation via the MAPK signaling pathway. Thus, combined administration of ICA with BMSCs has great potential in cartilage defect therapy.

PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein–coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin–WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration.

Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers

The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.