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Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
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1996 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 316, no 1, 137-142 p.Article in journal (Refereed) Published
Abstract [en]

In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

Place, publisher, year, edition, pages
1996. Vol. 316, no 1, 137-142 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-79473OAI: oai:DiVA.org:liu-79473DiVA: diva2:542602
Available from: 2012-08-02 Created: 2012-08-02 Last updated: 2017-12-07Bibliographically approved
In thesis
1. The role of calcium and calcium-regulated proteins in neutrophil phagocytosis
Open this publication in new window or tab >>The role of calcium and calcium-regulated proteins in neutrophil phagocytosis
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophil phagocytosis is an essential component of the innate immunity against invading pathogens. The two types of phagocytosis that are investigated in detail are IgG- and C3bi-mediated phagocytosis. Although the two types are controlled differently, they share the same driving force - reorganisation of the actin cytoskeleton. Subcellular elevations of intracellular free calcium concentration ([Ca2+]1), are critical for this kind of functional response within the neutrophils.

The aim of this study was to try to understand how calcium and certain calciumregulated proteins control phagocytosis in neutrophils, especially the remodelling of the actin cytoskeleton during pseudopod fonnation and the regulation of phagolysosome fusion.

By immunofluorescence staining (IF) and confocal microscopy, we analysed the distribution of Ca2+ stores using antibodies against Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase (SERCA2) and calreticulin, during phagocytosis. The results showed a distinct accumulation of Ca2+ stores around phagosomes and pseudopods. This accumulation is coherent with a local Ca2+ rise seen in the area of phagocytosis and provides a model for how this localised [Ca2+]i is regulated in neutrophils. To further investigate if inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores are involved, we analysed the subcellular distribution of IP3-receptors (IP3Rs), which are located on the ci+ stores. The IP3Rs translocated in a similar manner as did SERCA2 and calreticulin, indicating that the IP3-sensitive Ca2+ stores are involved.

During phagocytosis, an accumulation of phospholipid- and calcium-binding proteins, annexins, can be seen in the periphagosomal area. Several studies have demonstrated that certain annexins promote Ca2+-dependent contact between phospholipid vesicles and/or isolated neutrophil-specific granule membranes. This suggests that annexins, apart from being involved in vesicle aggregation and fusion, participate together with filamentous actin (F-actin) in phagolysosome formation, by establishing a connection between the phagosomal membrane and granule membranes prior to fusion. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. We have, therefore, investigated the role of gelsolin, which is a protein that severs Factin by binding to the barbed ends, and thereby inhibits further polymerisation. The results show that both annexin I and Ill, and gelsolin translocates to the area of phagocytosis, in a Ca2+ -independent manner, where they eo-localise with F-actin.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 40 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 619
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28584 (URN)13738 (Local ID)91-7219-576-2 (ISBN)13738 (Archive number)13738 (OAI)
Public defence
2000-03-17, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-08-02Bibliographically approved

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Stendahl, Olle

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