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P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Adamczyk M, Griffiths R, Dewitt S, Knäuper V, Aeschlimann D - J. Cell. Sci. (2015)

Bottom Line: Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement.A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity.These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

View Article: PubMed Central - PubMed

Affiliation: Matrix Biology & Tissue Repair Research Unit and Arthritis Research UK Biomechanics and Bioengineering Center of Excellence, College of Biomedical and Life Sciences, Cardiff University, Cardiff CF14 4XY, UK AdamczykM@Cardiff.ac.uk AeschlimannDP@Cardiff.ac.uk.

No MeSH data available.


Related in: MedlinePlus

Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca2+ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca2+ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.
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JCS175968F3: Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca2+ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca2+ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.

Mentions: In P2X7R-expressing cells, the prolonged increase in [Ca2+]i upon BzATP application was followed within 30 s by extensive cell blebbing as visualized by real-time microscopy (Fig. 3A, arrows). The term ‘blebbing’ is used here to describe formation of plasma membrane projections due to Rho-dependent actin reorganization that follow P2X7R activation (MacKenzie et al., 2001; Pfeiffer et al., 2004). This response is P2X specific. Stimulation of the parental cells, which express P2Y receptors, with ATP induced smaller transient oscillations in [Ca2+]i but no apparent morphological changes (Fig. 3A). This led us to speculate that TG2 externalization might be linked to membrane bleb formation, and we used GFP-tagged TG2 to monitor its redistribution in live cells. We confirmed that P2X7R activation triggered externalization of tagged TG2 similar to wild-type TG2 (Fig. 3B). Analysis by confocal microscopy revealed a clear ubiquitous cytoplasmic distribution for TG2–GFP (Fig. 3C). Upon P2X7R activation, TG2–GFP was rapidly translocated into membrane blebs, and freely re-localized to sites where new membrane protrusions formed (Fig. 3C, arrow). However, despite abundant bleb formation, careful reconstruction from image sequences revealed that these large membrane protrusions remained continuous with the plasma membrane and were eventually retracted by cells. We obtained similar results for N- and C-terminally tagged TG2 indicating that the position of the tag did not substantially alter protein localization. Although we were unable to directly visualize TG2 release, a noticeable reduction in fluorescence upon P2X7R activation indicated that the intracellular pool of TG2 was rapidly diminished, consistent with its relocation into the medium (Fig. 3B).Fig. 3.


P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Adamczyk M, Griffiths R, Dewitt S, Knäuper V, Aeschlimann D - J. Cell. Sci. (2015)

Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca2+ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca2+ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696497&req=5

JCS175968F3: Membrane blebs induced by P2X7R activation contain TG2. (A) P2X7R signaling induces rapid membrane blebbing. Fluo-4-AM-loaded P2X7R cells were stimulated with BzATP while acquiring fluorescence and phase-contrast images by real-time microscopy to visualize morphological changes and Ca2+ signaling simultaneously (top). Membrane blebs are indicated by arrows. ATP stimulation of parental cells induces oscillating Ca2+ signals but no overt morphological changes (bottom). Scale bar: 25 µm. (B,C) TG2 redistributes into membrane blebs. To confirm export of tagged TG2, TG2- (wild-type, WT) or TG2–GFP-expressing P2X7R cells were stimulated with 100 µM BzATP for 10 min, chased for 30 min in agonist-free medium, followed by analysis of conditioned media and cell extracts for TG2 by western blotting (B). To localize GFP-tagged TG2 during BzATP stimulation, real-time confocal microscopy was employed. Genesis of a membrane bleb is depicted (arrows), with an optical section of GFP fluorescence overlaid onto phase-contrast images to correlate morphological changes with changes in TG2 distribution (C). Scale bar: 10 µm.
Mentions: In P2X7R-expressing cells, the prolonged increase in [Ca2+]i upon BzATP application was followed within 30 s by extensive cell blebbing as visualized by real-time microscopy (Fig. 3A, arrows). The term ‘blebbing’ is used here to describe formation of plasma membrane projections due to Rho-dependent actin reorganization that follow P2X7R activation (MacKenzie et al., 2001; Pfeiffer et al., 2004). This response is P2X specific. Stimulation of the parental cells, which express P2Y receptors, with ATP induced smaller transient oscillations in [Ca2+]i but no apparent morphological changes (Fig. 3A). This led us to speculate that TG2 externalization might be linked to membrane bleb formation, and we used GFP-tagged TG2 to monitor its redistribution in live cells. We confirmed that P2X7R activation triggered externalization of tagged TG2 similar to wild-type TG2 (Fig. 3B). Analysis by confocal microscopy revealed a clear ubiquitous cytoplasmic distribution for TG2–GFP (Fig. 3C). Upon P2X7R activation, TG2–GFP was rapidly translocated into membrane blebs, and freely re-localized to sites where new membrane protrusions formed (Fig. 3C, arrow). However, despite abundant bleb formation, careful reconstruction from image sequences revealed that these large membrane protrusions remained continuous with the plasma membrane and were eventually retracted by cells. We obtained similar results for N- and C-terminally tagged TG2 indicating that the position of the tag did not substantially alter protein localization. Although we were unable to directly visualize TG2 release, a noticeable reduction in fluorescence upon P2X7R activation indicated that the intracellular pool of TG2 was rapidly diminished, consistent with its relocation into the medium (Fig. 3B).Fig. 3.

Bottom Line: Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement.A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity.These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

View Article: PubMed Central - PubMed

Affiliation: Matrix Biology & Tissue Repair Research Unit and Arthritis Research UK Biomechanics and Bioengineering Center of Excellence, College of Biomedical and Life Sciences, Cardiff University, Cardiff CF14 4XY, UK AdamczykM@Cardiff.ac.uk AeschlimannDP@Cardiff.ac.uk.

No MeSH data available.


Related in: MedlinePlus