scholarly journals Gelsolin Mediates Collagen Phagocytosis through a Rac-dependent Step

2004 ◽  
Vol 15 (2) ◽  
pp. 588-599 ◽  
Author(s):  
Pamela D. Arora ◽  
Michael Glogauer ◽  
Andras Kapus ◽  
David J. Kwiatkowski ◽  
Christopher A. McCulloch
Keyword(s):  
Actin Filaments ◽  
Calcium Ionophore ◽  
Initial Contact ◽  
Β1 Integrin ◽  
Wild Type ◽  
Collagen Binding ◽  
Calcium Dependent ◽  
Collagen Phagocytosis ◽  
Α2β1 Integrin

The role of gelsolin, a calcium-dependent actin-severing protein, in mediating collagen phagocytosis, is not defined. We examined α2β1 integrin-mediated phagocytosis in fibroblasts from wild-type (WT) and gelsolin knockout (Gsn-) mice. After initial contact with collagen beads, collagen binding and internalization were 60% lower in Gsn- than WT cells. This deficiency was restored by transfection with gelsolin or with β1 integrin-activating antibodies. WT cells showed robust rac activation and increased [Ca2+]i during early contact with collagen beads, but Gsn- cells showed very limited responses. Transfected gelsolin in Gsn- cells restored rac activation after collagen binding. Transfection of Gsn- cells with active rac increased collagen binding to WT levels. Chelation of intracellular calcium inhibited collagen binding and rac activation, whereas calcium ionophore induced rac activation in WT and Gsn- cells. We conclude that the ability of gelsolin to remodel actin filaments is important for collagen-induced calcium entry; calcium in turn is required for rac activation, which subsequently enhances collagen binding to unoccupied α2β1 integrins.

scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

2002 ◽  
Vol 115 (13) ◽  
pp. 2669-2678 ◽  
Author(s):  
Anna Gustavsson ◽  
Annika Armulik ◽  
Cord Brakebusch ◽  
Reinhard Fässler ◽  
Staffan Johansson ◽  
...  
Keyword(s):  
Marked Reduction ◽  
Yersinia Pseudotuberculosis ◽  
Host Cells ◽  
Β1 Integrin ◽  
Complex Structures ◽  
Wild Type ◽  
Similar Reduction ◽  
Focal Complex ◽  
Β1 Integrins

Invasin of Yersinia pseudotuberculosis binds to β1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the β1-integrin-mediated internalization of Yersinia, a β1-integrin-deficient cell line, GD25, transfected with wild-type β1A, β1B or different mutants of the β1A subunit was used. Both β1A and β1B bound to invasin-expressing bacteria, but only β1A was able to mediate internalization of the bacteria. The cytoplasmic region of β1A, differing from β1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

scholarly journals Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

1996 ◽  
Vol 135 (1) ◽  
pp. 153-167 ◽  
Author(s):  
J Peränen ◽  
P Auvinen ◽  
H Virta ◽  
R Wepf ◽  
K Simons
Keyword(s):  
Epithelial Cells ◽  
Membrane Transport ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Wild Type ◽  
Expression Systems ◽  
Vesicular Stomatitis

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

The Role of the cAMP-Response Element in the Bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2987-2987
Author(s):  
Hong Xiang ◽  
Linda M. Boxer
Keyword(s):  
B Cells ◽  
Wild Type ◽  
Camp Response Element ◽  
Response Element ◽  
Calcium Dependent ◽  
Flow Cytometric ◽  
Immature B Cells

Abstract We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.

scholarly journals RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

2004 ◽  
Vol 123 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Guangju Ji ◽  
Morris E. Feldman ◽  
Kai Su Greene ◽  
Vincenzo Sorrentino ◽  
Hong-Bo Xin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Wild Type ◽  
Calcium Dependent ◽  
Outward Currents ◽  
Associated Proteins ◽  
Kinetics Of ◽  
Spontaneous Transient Outward Currents

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

scholarly journals α2β1 integrin expression in the tumor microenvironment enhances tumor angiogenesis in a tumor cell–specific manner

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1980-1988 ◽  
Author(s):  
Zhonghua Zhang ◽  
Norma E. Ramirez ◽  
Thomas E. Yankeelov ◽  
Zhengzhi Li ◽  
Laura E. Ford ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Angiogenesis ◽  
Genetic Alterations ◽  
Wild Type ◽  
B16f10 Melanoma ◽  
B16f10 Cells ◽  
Low Levels ◽  
Α2β1 Integrin ◽  
Integrin Α2

To define the role of the α2β1 integrin in pathologic angiogenesis, we investigated tumor-associated growth and angiogenesis in wild-type and α2-null mice. Our findings reveal that the α2β1 integrin plays an important role in angiogenesis via regulation of VEGFR1 expression. When challenged with B16F10 melanoma cells, mice lacking α2β1 integrin ex-pression exhibit increased tumor angiogenesis associated with up-regulated VEGFR1 expression. In contrast, there was no α2β1 integrin-dependent difference in the angiogenic response to Lewis lung carcinoma (LLC) cells. Interestingly, whereas B16F10 cells secrete high levels of placental growth factor (PLGF), LLC cells produce high levels of VEGF, but low levels of PLGF. The α2β1 integrin-dependent difference in angiogenesis was restored to LLC cells by expression of PLGF, strongly suggesting that the angiogenic phenotype and tumor growth in the α2-null host is dependent on specific interactions between the tumor cell and the genetically defined integrin repertoire of the host microenvironment. Thus integrin α2-null mice represent an example of genetic alterations of “the soil” determining response to the “seed.”

scholarly journals Role of Flagellin and the Two-Component CheA/CheY System of Listeria monocytogenes in Host Cell Invasion and Virulence

2004 ◽  
Vol 72 (6) ◽  
pp. 3237-3244 ◽  
Author(s):  
Lone Dons ◽  
Emma Eriksson ◽  
Yuxuan Jin ◽  
Martin E. Rottenberg ◽  
Krister Kristensson ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Tumor Necrosis Factor Receptor ◽  
Soft Agar ◽  
Initial Contact ◽  
Wild Type ◽  
Chemotaxis Proteins ◽  
Deficient Mice ◽  
Necrosis Factor

ABSTRACT The flagellum protein flagellin of Listeria monocytogenes is encoded by the flaA gene. Immediately downstream of flaA, two genes, cheY and cheA, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in flaA. cheY, and cheA to elucidate their role in the biology of infection with L. monocytogenes. The ΔcheY, ΔcheA, and double-mutant ΔcheYA mutants, but not ΔflaA mutant, were motile in liquid media. However, the ΔcheA mutant had impaired swarming and the ΔcheY and ΔcheYA mutants were unable to swarm on soft agar plates, suggesting that cheY and cheA genes encode proteins involved in chemotaxis. The ΔflaA, ΔcheY, ΔcheA, and ΔcheYA mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the ΔflaA and ΔcheYA mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the ΔflaA or ΔcheYA mutant. In summary, we show that listerial flaA. cheY, and cheA gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that flaA could also be involved in the triggering of immune responses.

scholarly journals A novel PilR/PilS two-component system regulates NE pilus production in Clostridium perfringens

2021 ◽  
Author(s):  
Yuanyuan Zhou ◽  
Dion Lepp ◽  
Jason Carere ◽  
Hai Yu ◽  
Chengbo Yang ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Clostridium Perfringens ◽  
Binding Activity ◽  
Necrotic Enteritis ◽  
Wild Type ◽  
Two Component System ◽  
Collagen Binding ◽  
Component System ◽  
Two Component

Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS), designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS, in the regulation of pilin production. Isogenic pilR , agrB and virR null mutants were generated from parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1 pilR mutant, while production in its complement (CP1 pilR +) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type, but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I – V) than CP1 ( p ≤ 0.05), whereas this activity was restored in the complemented strain ( p > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 ( p > 0.05), whereas the complemented strains exhibited significantly reduced binding ( p ≤ 0.05). These data suggest that the PilRS TCS positively regulates pilus production in C. perfringens , while the Agr-like QS system may serve as a negative regulator of this operon. Importance Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the post-antibiotic era. Multiple factors in C. perfringens , including both virulent and non-virulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development and a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens . In addition, the Agr-like quorum sensing signalling pathway was found to be involved in the regulation. These findings have identified additional targets for developing non-antibiotic strategies to control NE disease.

The α2β1 Integrin Regulates Hematopoietic Stem Cell Engraftment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1328-1328
Author(s):  
Andrea M. Sheehan ◽  
Laura E. Ford ◽  
Daniel C. Link ◽  
Mary M. Zutter
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Quantitative Difference ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Ki 67 ◽  
Hematopoietic Stem ◽  
Α2β1 Integrin

Abstract The α2β1 integrin, a receptor for collagen, laminin, and other matrix and non-matrix ligands, is expressed on some subsets of hematopoietic stem cells (HSC). Previous studies of mobilized HSC suggested that α2β1 integrin expression may distinguish long-term (LT-HSC) from short-term (ST-HSC) HSC. We sought to address the role of the integrin in normal hematopoiesis and following bone marrow transplant. Evaluation of mice in which the α2β1 integrin was genetically deleted demonstrated that peripheral blood counts, bone marrow cellularity, and bone marrow morphology were identical in α2-null mice and their wild type littermate controls, suggesting that α2β1 integrin is not required for hematopoiesis. To further explore the role of the α2β1 integrin in HSC homing and engraftment, we undertook a competitive repopulation assay using a 1:1 mix of α2β1−/− (Ly5.2) and α2β1+/+ (Ly5.1) cells transplanted into lethally irradiated Ly5.1/Ly5.2 mice. Analysis of peripheral blood, bone marrow, spleen, and lymph nodes post-transplant by FACS for the detection of lineage markers (CD3, B220, CD11b, F4/80, Gr-1, NK1.1) and for expression of either CD45.1 (Ly5.1) or CD45.2 (Ly5.2), demonstrated significantly greater reconstitution with α2β1−/− marrow than α2β1+/+ wild type marrow for lymphoid, myeloid, and monocytic lineages. The ratios ranged from 2:1(CD3, p=0.0024) − 5.6:1 (F4/80, p=0.0021). Secondary transplants of bone marrow harvested from primarily reconstituted mice showed similar findings with α2β1−/− (Ly5.2) to α2β1+/+ (Ly5.1) ratios ranging from 3.2:1 (CD3, p=0.0001) to 6.5:1 (F4/80, p<0.0001). The ratio was also altered within the HSC population (Lin−ThyloSca+c-kit+ (LTSK)) after transplant with an α2β1−/− (Ly5.2) to α2β1+/+ (Ly5.1) ratio of 4.63:1 (p=0.0136). To evaluate the molecular mechanisms that resulted in dramatic engraftment advantage, quantitative analysis of resting HSC in α2-null and wild-type bone marrow and spleen by FACS was performed. However, no quantitative difference in the LTSK population, and no difference in LT-HSC (LSK CD34−Flk-2−), ST-HSC (LSK CD34+Flk-2−), or multipotent progenitors (LSK CD34+ Flk-2+) was identified. FACS analysis of α4β1 expression showed no increase in α4β1 expression in a2-null HSC compared to wild-type, indicating that α2-null HSC do not compensate with upregulation of α4β1 expression. Homing assays using CFSE labeled α2-null or wild-type bone marrow transplanted into lethally irradiated α2-null or wild type recipients showed no difference in homing, regardless of the donor or recipient genotype. Although HSC numbers were the same, methylcellulose cultures for hematopoietic progenitors showed significantly greater numbers of CFU-C for α2-null bone marrow compared to wild-type (p=0.0067), suggesting either a proliferative advantage or heightened sensitivity to growth factors present in the media. Immunohistochemical analysis of bone marrow from α2-null and wild-type mice for Ki-67 showed significantly greater proliferation in the wild-type compared to α2-null bone marrow. These studies suggest that although α2β1 integrin is not required for normal hematopoiesis, the integrin may play important roles in regulation of HSC proliferation as well as maintenance of the HSC niche.

Role of murine integrin α2β1 in thrombus stabilization and embolization: Contribution of thromboxane A2

2007 ◽  
Vol 98 (11) ◽  
pp. 1072-1080 ◽  
Author(s):  
Miroslava Pozgajova ◽  
Judith Cosemans ◽  
Imke Munnix ◽  
Beate Eckes ◽  
Bernhard Nieswandt ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Thromboxane A2 ◽  
Wild Type ◽  
Thrombosis Model ◽  
Thromboxane Receptors ◽  
Thromboxane A2 Analogue ◽  
Α2β1 Integrin

SummaryPlatelets stably interact with collagen via glycoprotein (GP)VI and α2β1 integrin. With α2-null mice, we investigated the role of α2β1 in thrombus formation and stability in vivo and in vitro. Using a FeCl3-induced thrombosis model, in arteries from α2-null mice smaller thrombi were formed with more embolization compared to vessels from wild-type mice. Aspirin treatment of wild-type mice causes similar effects, while the thromboxane A2 analogue U46619 was borderline effective in suppressing the embolisation in α2-null mice. In vitro, perfusion of α2-null blood over collagen resulted in formation of thrombi that were smaller and looser in appearance, regardless of the presence or absence of coagulation. Aspirin treatment or blockage of thromboxane receptors provoked embolus formation in wildtype blood, while U46619 normalized thrombus formation in blood from α2-null mice. We conclude that integrin α2β1 plays a role in stabilizing murine thrombi, likely by enhancing GPVI activation and thromboxane A2 release. The increased embolization in α2-null mice may argue against the use of α2β1 integrin inhibitors for antithrombotic therapy.

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