79 research outputs found
EM Structure of the Ectodomain of Integrin CD11b/CD18 and Localization of Its Ligand-Binding Site Relative to the Plasma Membrane
One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMÎČ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation
Stress Hematopoiesis Is Regulated by the KrĂŒppelâLike Transcription Factor ZBPâ89
Previous studies have shown that ZBPâ89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBPâ89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89 â / â HSPC also resulted in a myeloidâtoâB lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89 â / â HSPC are reproduced in vitro in the ZBPâ89 âsilenced multipotent hematopoietic cell line FDCPâMix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATAâ1 in ZBP89âdeficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBPâ89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATAâ1 . These data identify an important role for ZBPâ89 in regulating stress hematopoiesis in adult mouse bone marrow. S tem C ells 2014;32:791â801Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106141/1/stem1598.pd
Recommended from our members
A microfluidic renal proximal tubule with active reabsorptive function
In the kidney, the renal proximal tubule (PT) reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity
Recommended from our members
Structural basis for pure antagonism of integrin αVÎČ3 by a high affinity form of fibronectin
Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVÎČ3 bound to a physiologic ligand: the 10th type III RGD-domain of wild-type fibronectin (wtFN10), or to a high affinity mutant (hFN10) that acts as a pure antagonist. Comparison of these structures revealed a central Ï - Ï interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the ÎČ3-subunit that blocked conformational changes triggered by wtFN10, and trapped hFN10-bound αVÎČ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side-chains, or reorienting Trp1496 away from Tyr122, converted hFN10 into a partial agonist. The findings offer new insights on the mechanism of integrin activation and a basis for design of RGD-based pure antagonists
N-terminal sequence of human leukocyte glycoprotein Mol: conservation across species and homology to platelet IIb/IIIa
Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25944/1/0000006.pd
Recommended from our members
Collective Epithelial Migration Drives Kidney Repair after Acute Injury
Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidneyâs remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI
Modulation of surface CD11/CD18 glycoproteins (Mo1, LFA-1, p150,95) by human mononuclear phagocytes
Mo1, LFA-1, and p150,95 are structurally related glycoproteins of the CD11/CD18 complex that are expressed on the membrane of human leukocytes. In the neutrophil, the surface expression of the CD11/CD18 complex is up-modulated (Mo1 > p150,95 >> LFA-1) by stimulatory factors that include calcium ionophore A23187, phorbol myristate acetate (PMA), and N--formyl--leucyl--phenylalanine (fMLP). Here, in an immunofluorescence analysis, we have examined CD11/CD18 glycoprotein expression by human monocytes, pulmonary alveolar macrophages (PAM, obtained by bronchoalveolar lavage), and breast milk macrophages (BMM) as compared to neutrophils before and after exposure to A23187 (1 [mu]M), fMLP (0.1 [mu]M), or PMA (0.1 [mu]g/ml) ft 37[deg]C. Unstimulated monocytes within unfractionated blood mononuclear cells kept at 4[deg]C (n = 13) expressed all three CD11/CD18 glycoproteins, and exposure to A23187 resulted in significant increases in the surface expression of Mol (median of 5.7-fold), LFA-1 (median of 2.1-fold), and p150,95 (median of 7.2-fold). Exposure to fMLP- or PMA-stimulated increases of lesser magnitude. CD11/CD18 expression by PAM (n = 9) was barely detectable and was unaffected by exposure to A23187. In contrast, BMM (n = 11) expressed all three CD11/CD18 glycoproteins (with considerable variability among specimens), but no increase was stimulated by A23187. These results demonstrate that monocytes, like neutrophils, have the capacity to respond to activating factors with an increase in CD11/CD18 glycoprotein expression; macrophage differentiation is accompanied by a loss (PAM) or retention (BMM) of CD11/CD18 expression that is unmodulated in response to activation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27416/1/0000453.pd
Recommended from our members
Crystal structure of the complete integrin αVÎČ3 ectodomain plus an α/ÎČ transmembrane fragment
We determined the crystal structure of 1TM-αVÎČ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and ÎČ3 subunits. 1TM-αVÎČ3 is more compact and less active in solution when compared with ÎTM-αVÎČ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the αâÎČ interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVÎČ3 on live cells yields a donorâmembrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2âthigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state
Molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in Baghdad city - Iraq
Background: Although G6PD deficiency is the most common genetically determined blood disorder among Iraqis, its molecular basis has only recently been studied among the Kurds in North Iraq, while studies focusing on Arabs in other parts of Iraq are still absent. Methods: A total of 1810 apparently healthy adult male blood donors were randomly recruited from the national blood transfusion center in Baghdad. They were classified into G6PD deficient and non-deficient individuals based on the results of methemoglobin reduction test (MHRT), with confirmation of deficiency by subsequent enzyme assays. DNA from deficient individuals was studied using a polymerase chain reaction-Restriction fragment length polymorphism (PCR-RFLP) for four deficient molecular variants, namely G6PD Mediterranean (563 CÂźT), Chatham (1003 GÂźA), A- (202 GÂźA) and Aures (143 TÂźC). A subset of those with the Mediterranean variant, were further investigated for the 1311 (CÂźT) silent mutation. Results: G6PD deficiency was detected in 109 of the 1810 screened male individuals (6.0%). Among 101 G6PD deficient males molecularly studied, the Mediterranean mutation was detected in 75 cases (74.3%), G6PD Chatham in 5 cases (5.0%), G6PD A- in two cases (2.0%), and G6PD Aures in none. The 1311 silent mutation was detected in 48 out of the 51 G6PD deficient males with the Mediterranean variant studied (94.1%). Conclusions: Three polymorphic variants namely: the Mediterranean, Chatham and A-, constituted more than 80% of G6PD deficient variants among males in Baghdad. Iraq. This observation is to some extent comparable to othe
- âŠ