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First published online 21 March 2006
doi: 10.1242/jcs.02872

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Stimulation of G_q-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK

¹ University of Leeds, Institute of Membrane and Systems Biology, Garstang Building, Mount Preston Street, Leeds LS2 9JT, UK
² Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK
³ Department of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520, USA

View larger version (31K): [in a new window]   Fig. 1. Endogenous II spectrin is a component of the Gq/11-associated protein complex. (A) RT-PCR. Both wild-type (WT, lane 1) and M1-CHO cells (M1, lane 2) express II spectrin transcripts. No protein was detected in the samples without reverse transcription (RT: lane 4 and 5) and without vector (lane 3). (B) Immunoprecipitation assay. Full-length II spectrin (240 kDa) is present in the Gq/11-associated protein complex isolated from M1-CHO cells, but not in the immunocomplex incubated with the control antibody anti-Myc.

View larger version (101K): [in a new window]   Fig. 2. II spectrin colocalizes with Gq/11 at the periphery of M1-CHO cells. (A) Expression of the surface marker CD8 (red) is at the plasma membrane, whereas YFP-II spectrin (green) is distributed in the cytosol with enrichment under the plasma membrane (merge). YFP-II spectrin is not expressed in the second cell in this field because of its low transfection efficiency (maximum 30%) compared with CD8 (80-90%). (B) Endogenous Gq/11 (red) is distributed throughout the cytoplasm and colocalization with YFP-II spectrin (green) is seen at the membrane skeleton (merge). (C) Myc-tagged Gq (red) similarly colocalizes with YFP-II spectrin (green) at the plasma membrane skeleton (merge). Insets in merged images show higher magnification views of the boxed region of the main images. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 3. Reversible redistribution of II spectrin is a downstream event of M1 mAChR activation. Redistribution of YFP-II spectrin starts approximately 42 seconds after Oxo-M application and lasts for 370 seconds in M1-CHO cells. II spectrin redistributes into the bleb-ends (arrows) during blebbing and reverts to the pre-activation positions (arrowheads) when stabilized. For the live image of this cell, see supplementary material Movie 1. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 4. PLC mediates muscarinic agonist-induced II spectrin redistribution. Digital deconvolved images of a M1-CHO cell expressing YFP-II spectrin (green) were taken at 0 and 250 seconds after Oxo-M application. Inhibition of PLC activity by U73122 abolishes both redistribution of YFP-II spectrin and membrane blebbing. Bar, 10 µm.

View larger version (11K): [in a new window]   Fig. 5. Muscarinic agonist-induced spectrin remodeling occurs subsequent to a rise in [Ca2+]i. Activation of M1 mAChRs causes a transient increase in [Ca2+]i in M1-CHO cells. This rapid rise in [Ca2+]i (<20 seconds) precedes redistribution of YFP-II spectrin, and spectrin redistribution (solid bar on the top indicates duration) continues after [Ca2+]i returned to the basal levels. The graph represents a typical response of Fura-2AM-loaded M1-CHO cells, and the average duration of spectrin redistribution (n=12).

View larger version (31K): [in a new window]   Fig. 6. Ionomycin can trigger redistribution of II spectrin but lacks a reversible mechanism. II spectrin redistributes into the bleb ends (arrows) upon an application of ionomycin. After ionomycin-induced membrane blebbing, II spectrin loses its assembly at the membrane skeleton and diffuses into the cytosol (arrowheads). For the live image of this cell, see supplementary material Movie 2. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 7. Muscarinic agonist-induced spectrin redistribution depends on transient activation of ROCK. (A) Proteolytic conversion from the full-length (*) to C-terminally cleaved, active ROCK (**) is not significantly induced in M1-CHO cells stimulated with 5 µM Oxo-M for 5 minutes. (B) Transient expression of Flag-tagged kinase-dead (KD) ROCK-II (top row, red) diminishes Oxo-M-induced membrane blebbing and redistribution of YFP-II spectrin (top row, green). Images were taken 3 minutes after activation of M1 mAChRs. Flag-tagged constitutively active (CA) ROCK-II (bottom row, red) causes intense membrane blebbing accompanied with cell rounding in the absence of M1 mAChR activation. Images were taken 0 minutes after activation of M1 mAChRs. (C) Transient expression of Myc-tagged dominant-negative Rho (RhoA N19, top row, red) has little effects on Oxo-M-induced redistribution of YFP-II spectrin to the bleb end (top row, green, arrows). Images were taken 3 minutes after activation of M1 mAChRs. Overexpression of Myc-tagged constitutively active RhoA (Rho V14, bottom row, red) itself causes neither membrane blebbing nor cell rounding. Images were taken 0 minutes after activation of M1 mAChRs. Bar, 10 µm.

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