Recycling and LFA‐1‐dependent trafficking of ICAM‐1 to the immunological synapse

Little is known about how adhesion molecules on APCs accumulate at immunological synapses. We show here that ICAM‐1 on APCs is continuously internalized and rapidly recycled back to the interface after antigen‐priming T‐cell contact. The internalization rate is high in APCs, including Raji B cells and dendritic cells, but low in endothelial cells. Internalization is significantly reduced by inhibitors of Na⁺/H⁺ exchangers (NHEs), suggesting that members of the NHE‐family regulate this process. Once internalized, ICAM‐1 is co‐localized with MHC class II in the polarized recycling compartment. Surprisingly, not only ICAM‐1, but also MHC class II, is targeted to the immunological synapse through LFA‐1‐dependent adhesion. Cytosolic ICAM‐1 is highly mobile and forms a tubular structure. Inhibitors of microtubule or actin polymerization can reduce ICAM‐1 mobility, and thereby block accumulation at immunological synapses. Membrane ICAM‐1 also moves to the T‐cell contact zone, presumably through an active, cytoskeleton‐dependent mechanism. Collectively, these results demonstrate that ICAM‐1 can be transported to the immunological synapse through the recycling compartment. Furthermore, the high‐affinity state of LFA‐1 on T cells is critical to induce targeted movements of both ICAM‐1 and MHC class II to the immunological synapse on APCs. J. Cell. Biochem. 111: 1125–1137, 2010. © 2010 Wiley‐Liss, Inc.     

Leukocyte Migration in Adipose Tissue of Mice Null for ICAM‐1 and Mac‐1 Adhesion Receptors

Objective: To determine whether the leukocyte adhesion receptors ICAM‐1 and Mac‐1, regulators of immune cell migration, have an intrinsic role within adipose tissue by 1) analyzing the expression of ICAM‐1 in adipose tissue, 2) identifying leukocyte populations within adipose tissue, and 3) determining whether ICAM‐1 and Mac‐1 mutant mice exhibit abnormal numbers of adipose tissue leukocytes. Research Methods and Procedures: Wild‐type, ICAM‐1^?/?, and Mac‐1^?/? mice were fed a long‐term high‐fat diet. ICAM‐1 expression was analyzed by Northern blot and immunohistochemistry. Leukocytes within adipose tissue were identified by immunohistochemistry and flow cytometry. Results: ICAM‐1 was expressed in adipose tissue and localized to the vascular endothelium. Macrophages and lymphocytes were prevalent within the stromal‐vascular cell fraction of adipose tissue, and gender‐specific differences were observed, with adipose tissue from female mice containing significantly more macrophages than tissue from male mice. Numbers of leukocytes in ICAM‐1^?/? and Mac‐1^?/? mice were not different from wild‐types, however, indicating that these adhesion receptors are not required for leukocyte migration into adipose tissue. Discussion: Our results documented leukocyte populations within adipose tissue, which may be involved in the development of heightened inflammation that is characteristic of obesity.     

Genetic correction of Werner syndrome gene reveals impaired pro‐angiogenic function and HGF insufficiency in mesenchymal stem cells

WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN^+/+ mesenchymal stem cells (MSCs) exhibited improved pro‐angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN^?/? MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN^?/? MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN^+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro‐angiogenic function of WS‐MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A novel role for acid‐sensing ion channels in Pavlovian reward conditioning

Pavlovian fear conditioning has been shown to depend on acid‐sensing ion channel‐1A (ASIC1A); however, it is unknown whether conditioning to rewarding stimuli also depends on ASIC1A. Here, we tested the hypothesis that ASIC1A contributes to Pavlovian conditioning to a non‐drug reward. We found effects of ASIC1A disruption depended on the relationship between the conditional stimulus (CS) and the unconditional stimulus (US), which was varied between five experiments. In experiment 1, when the CS preceded the US signaling an upcoming reward, Asic1a^?/? mice exhibited a deficit in conditioning compared to Asic1a^+/+ mice. Alternatively, in experiment 2, when the CS coinitiated with the US and signaled immediate reward availability, the Asic1a^?/? mice exhibited an increase in conditioned responses compared to Asic1a^+/+ mice, which contrasted with the deficits in the first experiment. Furthermore, in experiments 3 and 4, when the CS partially overlapped in time with the US, or the CS was shortened and coinitiated with the US, the Asic1a^?/? mice did not differ from control mice. The contrasting outcomes were likely because of differences in conditioning because in experiment 5 neither the Asic1a^?/? nor Asic1a^+/+ mice acquired conditioned responses when the CS and US were explicitly unpaired. Taken together, these results suggest that the effects of ASIC1A disruption on reward conditioning depend on the temporal relationship between the CS and US. Furthermore, these results suggest that ASIC1A plays a critical, yet nuanced role in Pavlovian conditioning. More research will be needed to deconstruct the roles of ASIC1A in these fundamental forms of learning and memory.     

Lifelong reduction in complex IV induces tissue‐specific metabolic effects but does not reduce lifespan or healthspan in mice

Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1^?/? mice. The lack of deleterious phenotypes in Surf1^?/? mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1^?/? vs. Surf1^+/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1^+/+ and Surf1^?/? mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1^+/+ and Surf1^?/? mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1^?/? adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1^?/? mice. Finally, mitochondrial unfolded protein response (UPR^mt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPR^mt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1^?/? mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.     

Quantitative proteome‐based systematic identification of SIRT7 substrates

SIRT7 is a class III histone deacetylase that is involved in numerous cellular processes. Only six substrates of SIRT7 have been reported thus far, so we aimed to systematically identify SIRT7 substrates using stable‐isotope labeling with amino acids in cell culture (SILAC) coupled with quantitative mass spectrometry (MS). Using SIRT7^+/+ and SIRT7 ^?/? mouse embryonic fibroblasts as our model system, we identified and quantified 1493 acetylation sites in 789 proteins, of which 261 acetylation sites in 176 proteins showed ≥2‐fold change in acetylation state between SIRT7^?/? and SIRT7^+/+ cells. These proteins were considered putative SIRT7 substrates and were carried forward for further analysis. We then validated the predictive efficiency of the SILAC–MS experiment by assessing substrate acetylation status in vitro in six predicted proteins. We also performed a bioinformatic analysis of the MS data, which indicated that many of the putative protein substrates were involved in metabolic processes. Finally, we expanded our list of candidate substrates by performing a bioinformatics‐based prediction analysis of putative SIRT7 substrates, using our list of putative substrates as a positive training set, and again validated a subset of the proteins in vitro. In summary, we have generated a comprehensive list of SIRT7 candidate substrates.     

Deletion of junctional adhesion molecule A from platelets increases early‐stage neointima formation after wire injury in hyperlipidemic mice

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM‐A) was recently described to regulate platelet activation. Specific deletion of JAM‐A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet‐derived JAM‐A to neointima formation after vascular injury. Mice with or without platelet‐specific (tr)JAM‐A‐deficiency in an apolipoprotein e (apoe^?/?) background underwent wire‐induced injury of the common carotid artery. Ex vivo imaging by two‐photon microscopy revealed increased platelet coverage at the site of injury in trJAM‐A‐deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM‐A‐deficient platelets than to control platelets. Inhibition of α_Mβ₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM‐A^?/? apoe^?/? mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM‐A^+/+ apoe^?/? littermates 2 weeks, but not 4 weeks after injury. Re‐endothelialization was decreased in trJAM‐A^?/? apoe^?/? mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM‐A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.     

Expression of PD‐1/LAG‐3 and cytokine production by CD4+ T cells during infection with Plasmodium parasites

CD4⁺ T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4⁺ T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4⁺ T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD‐1 and LAG‐3, as early as 6 days after infection, whereas those from either Listeria monocytogenes‐ or Leishmania major‐infected mice did not. In response to T‐cell receptor stimulation, CD4⁺ T cells from mice infected with all the pathogens under study produced high concentrations of IFN‐γ. IL‐2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD‐1 and its ligands resulted in increased IFN‐γ production in response to Plasmodium antigens, implying that PD‐1 expressed on activated CD4⁺ T cells actively inhibits T cell immune responses. Studies using Myd88^?/?, Trif^?/? and Irf3^?/? mice showed that induction of these CD4⁺ T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD‐1 and LAG‐3 on CD4⁺ T cells and their reduced IL‐2 production are common characteristic features of Plasmodium infection.

     

Enhanced microglial pro‐inflammatory response to lipopolysaccharide correlates with brain infiltration and blood–brain barrier dysregulation in a mouse model of telomere shortening

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self‐renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as ‘priming’. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first‐generation G1 mTerc^?/?)‐ and late‐generation (third‐generation G3 and G4 mTerc^?/?) telomerase‐deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late‐generation mTerc^?/? microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc^?/? microglia are comparable with microglia derived from G1 mTerc^?/? mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc^?/? microglia mice show an enhanced pro‐inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age‐associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood–brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.     

Involvement of caspase‐1 in inflammasomes activation and bacterial clearance in S. aureus‐infected osteoblast‐like MG‐63 cells

Staphylococcus aureus, a versatile Gram‐positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase‐1 that proteolytically matures and promotes the secretion of mature IL‐1β and IL‐18. The role of inflammasomes and caspase‐1 in the secretion of mature IL‐1β and in the defence of S. aureus‐infected osteoblasts has not yet been fully investigated. We show here that S. aureus‐infected osteoblast‐like MG‐63 but not caspase‐1 knock‐out CASP1 ^?/?MG‐63 cells, which were generated using CRISPR‐Cas9 technology, activate the inflammasome as monitored by the release of mature IL‐1β. The effect was strain‐dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes‐related IL‐1β production. Furthermore, we found that the lack of caspase‐1 in CASP1 ^?/?MG‐63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 ^?/? MG‐63 compared to wild‐type cells. Our results demonstrate that osteoblast‐like MG‐63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase‐1 in bacterial clearance.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

NMR characterization of the conformational fluctuations of the human lymphocyte function‐associated antigen‐1 I‐domain

Lymphocyte function‐associated antigen‐1 (LFA‐1) is an integrin protein that transmits information across the plasma membrane through the so‐called inside‐out and outside‐in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA‐1 I‐domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model‐free analysis. We then extended this analysis to the millisecond timescale motions by measuring ¹⁵N‐¹H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I‐domain using their corresponding X‐ray crystallographic structures. Our results highlight the importance of the low‐frequency motions of the LFA‐1 I‐domain in the inactive apo‐state. We found in particular that α‐helix 7 is in a position in the apo‐closed state that cannot be fully described by any of the existing X‐ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA‐1 I‐domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α‐helix 7 that is required for the activation of LFA‐1.     

Antidepressant‐like effects and basal immobility depend on age and serotonin transporter genotype

Monoamine uptake inhibitors are common treatments for depression; however, the therapeutic efficacy of these drugs varies widely. Two factors that are commonly linked to clinical outcome are age and serotonin transporter (SERT) genotype. Mouse models provide powerful tools to study consequences of age and genotype on antidepressant‐like efficacy; however, to date, systematic studies of this nature are lacking. Here, we used the tail suspension test (TST), a preclinical assay for antidepressant efficacy, to gain insight into age and SERT genotype dependency of immobility time in the TST under control conditions (saline injection) and in response to the tricyclic antidepressant, desipramine (DMI). Immobility after saline injection in juvenile, adolescent, adult, mature adult and middle‐aged mice (postnatal days 21, 28, 90, 210 and 300, respectively) significantly increased with age; however, the rate of increase was slower for SERT null (?/?) mice than for wild‐type (+/+) or heterozygote (+/?) mice. Desipramine reduced immobility across ages and SERT genotypes. Middle‐aged, but not adult, SERT^?/? mice were significantly more sensitive to DMI than age‐matched SERT^+/+ or SERT^+/? mice. Desipramine was less potent in middle‐aged SERT^+/+ and SERT^+/? mice than in adult SERT^+/+ or SERT^+/? mice. Regardless of age, DMI's maximal effects were greater in SERT^?/? mice than in SERT^+/+ or SERT^+/? mice. These results show that immobility time in the TST varies as a function of age and SERT genotype, underscoring the utility of the TST as a potential model to examine age‐ and SERT genotype‐dependent influences on antidepressant response.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Spatial mapping of affinity changes for the integrin LFA‐1 during cell migration using clusters identified based on local density

Localization microscopy methods like Stochastic Optical Reconstruction Microscopy (STORM) are very well suited for exploring clustering of proteins, as the data inherently provide a list of molecular coordinates. Here we use state‐of‐art cluster analysis algorithms (DBSCAN) to explore the clustering behaviour of different affinity forms of the integrin LFA‐1. It has been suggested that LFA‐1 may form clusters, in order to increase the avidity to ICAM‐1. However, this hypothesis still seems to be controversial. In this study, we found, variations in clustering behaviour among the different affinity forms of LFA‐1 in migrating T‐cells. We found that panLFA‐1 is located in clusters throughout the polarised cell on ICAM‐1, with an increased density of molecules and clusters in the mid area and rear of the cell, whereas the intermediate and high affinity form of LFA‐1 showed an increased number in the mid area of a migrating cell and the high affinity form of LFA‐1 in the front and rear. Together, these data suggest that, in addition to LFA‐1 conformation, protein clustering might play a role in controlling cell‐substrate adhesion on ICAM‐1.By applying the cluster analysis algorithm DBSCAN to localization microscopy data, integrin clusters could be identified and different cluster parameters could be quantified.      

Ca2+‐mediated exocytosis of subtilisin‐like protease 1: a key step in egress of Plasmodium falciparum merozoites

Egress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood‐stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra‐erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca²⁺ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca²⁺ just before egress, observed by time‐lapse video microscopy, suggested a role for intracellular Ca²⁺ in this process. Chelation of intracellular Ca²⁺ with chelators such as BAPTA‐AM or inhibition of Ca²⁺ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca²⁺ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin‐like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.     

BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinase

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3^+/+ and Tgfbr3^?/? mouse embryos, we show that Tgfbr3^?/? epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3^?/? epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3^+/+ cells; BMP2 induces cellular migration to Tgfbr3^+/+ levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3^?/? migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3^?/? epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.