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First published online 4 January 2005
doi: 10.1242/jcs.01625

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SH3 domain of spectrin participates in the activation of Rac in specialized calpain-induced integrin signaling complexes

¹ Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA
² Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Hiroshwa, Wako-shi Saitama 351-01, Japan
³ Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA

View larger version (41K): [in a new window]   Fig. 1. Spectrin colocalizes with integrin in clusters at the periphery of newly adherent BAE cells but has a more diffuse distribution in spreading cells. BAE cells were allowed to spread on fibronectin-coated coverslips for 30 minutes (A), 1 hour or 8 hours (B), fixed, permeabilized and dual-labeled with monoclonal antibody against vß3 integrin and polyclonal antibodies against spectrin. (A) Three independent fields of cells are shown and cells containing integrin clusters are indicated with arrows. (B) Focal adhesions are indicated with arrows. Bar, 20 µm.

View larger version (107K): [in a new window]   Fig. 2. Spectrin is localized in specialized ß3-integrin clusters that mediate the initial adhesion of BAE cells but is not present in focal complexes or focal adhesions. BAE cells were allowed to spread on fibronectin-coated coverslips fixed, permeabilized, and dual-labeled with monoclonal antibody against vß3 integrin and polyclonal antibodies against spectrin. Confocal analysis was performed on horizontal (x-y plane) optical sections (0.3 µm intervals). (A) Seven sections going from the upper to the lower surface of the cell (top panels to bottom panels) are shown. (B and C) Six sections going from the upper to the lower surface of the cell (top left to bottom right panel) are shown. Panel A shows sections through two cells 30 minutes after plating; Panel B, shows sections through a peripheral region of a cell 1 hour after plating; Panel C shows sections through a peripheral region of a cell 8 hours after plating. Bar, 5 µm.

View larger version (32K): [in a new window]   Fig. 3. Integrin signaling is required for spectrin clustering. BAE cells were allowed to spread on poly-L-lysine-coated coverslips in the presence of 2% serum for 30 minutes, fixed, permeabilized, and stained with monoclonal antibody against vß3 integrin and polyclonal antibodies against spectrin. Actin filaments were detected with TRITC-labeled phalloidin. Bar, 20 µm.

View larger version (52K): [in a new window]   Fig. 4. Formation of spectrin-containing clusters is dependent on calpain activity. BAE cells were detached and replated on fibronectin-coated coverslips in the presence of calpeptin (100 µg/ml) or MDL (150 µM) for 30 minutes, 1 hour or 8 hours, fixed, permeabilized and stained with polyclonal antibodies against spectrin and monoclonal antibody against vß3 integrin. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 5. Spectrin is cleaved by calpain upstream of cluster formation. BAE cells were serum-starved for 16 hours and either solubilized in SDS-containing buffer or allowed to spread on fibronectin-coated dishes for 15, 20 or 30 minutes. (A) Cells were solubilized in SDS-containing buffer and analyzed on western blots with polyclonal antibodies against bovine - and ß-spectrin. (B) BAE cells were allowed to spread on fibronectin-coated coverslips for 30 minutes or 8 hours, fixed and permeabilized. In the top panel, samples were incubated with polyclonal antibodies that detect only a 150 kD calpain-cleaved fragment of -spectrin and monoclonal antibody against calpain (as a marker for the early integrin-containing clusters). In the bottom panel, samples were incubated with antibodies against the calpain-generated fragment of spectrin and with antibodies against vinculin, a marker of focal complexes and focal adhesions. (C) Cells were fixed, permeabilized, and incubated with polyclonal antibodies that detect only the 150 kD calpain-cleaved fragment of spectrin. Arrows indicate spectrin-fragment-containing clusters. (D) Cells were plated for 20 or 30 minutes, fixed, permeabilized and stained with polyclonal antibodies specific for the 150 kD fragment of -spectrin. Confocal analysis was performed on horizontal (x-y plane) optical sections (0.3 µm intervals); for each time point, four sections going from the upper to the lower surface (left to right) of a single cell are shown. Arrows indicate spectrin-fragment-containing clusters. Bars, 20 µm (A-C); 5 µm (D).

View larger version (97K): [in a new window]   Fig. 6. Spectrin-containing integrin clusters form upstream of Rac activation. BAE cells were transiently transfected with plasmid encoding myc-tagged dominant-negative Rac (N17Rac). Cells were detached and replated on fibronectin for 1 hour, fixed and permeabilized. Spectrin distribution was detected with polyclonal antibodies against spectrin. Cells expressing dominant-negative Rac were detected with anti-myc antibody. Actin filaments were detected with TRITC-labeled phalloidin. Rac- and spectrin-containing clusters are indicated with arrows. Three independent fields are shown. Bar, 20 µm.

View larger version (56K): [in a new window]   Fig. 7. SH3 domain of spectrin delays cell spreading on fibronectin. BAE cells were transiently transfected with plasmid encoding HA epitope-tagged SH3 domain of spectrin or HA epitope-tagged SH3 domain of Crk. Cells were detached and either solubilized in SDS for analysis of SH3 domain expression on western blots (A) or replated on fibronectin-coated coverslips for 1 or 3 hours, fixed and permeabilized (B,C). Cells were stained with TRITC-labeled phalloidin to detect actin filaments. Transfected cells were detected with polyclonal antibodies against HA epitope. (B) Cells transfected with spectrin SH3 domain. (C) Cells transfected with Crk SH3 domain. Arrows indicate transfected cells. Bar, 20 µm.

View larger version (59K): [in a new window]   Fig. 8. SH3 domain of spectrin accumulates in the early integrin clusters. BAE cells were transiently transfected with plasmid encoding HA epitope-tagged SH3 domain. Cells were detached and replated on fibronectin for 1 hour, fixed and permeabilized. Cells were stained with monoclonal antibody against HA epitope and polyclonal antibodies against an autolytic fragment of calpain. (B) Confocal analysis was performed on horizontal (x-y plane) optical sections (0.3 µm intervals); three sections going from the upper to the lower region (left to right) of a single cell are shown. Arrows indicate calpain-fragment- and SH3-domain-containing clusters. Bar, 20 µm (A); 5 µm (B).

View larger version (29K): [in a new window]   Fig. 9. Constitutively active Rac overcomes the effect of the SH3 domain on cell spreading. BAE cells were transiently transfected with plasmid encoding EGFP-tagged SH3 domain alone or together with constitutively active Q61LRac. Cells were detached, washed and sorted. (A) Enriched populations of cells expressing EGFP-tagged SH3 domain alone were replated on fibronectin for 1 hour. (B) Enriched populations expressing SH3 domain together with constitutively active Q61LRac were replated on fibronectin for 20, 30, 45 minutes or 1 hour. Cells were fixed, permeabilized and stained with antibody against HA epitope to detect constitutively active Rac. Actin filaments were detected with TRITC-labeled phalloidin. Bar, 20 µm.

View larger version (49K): [in a new window]   Fig. 10. SH3 domain of spectrin participates in Rac activation. (A) CHO cells expressing human IIbß3 integrin were transiently transfected with HA-tagged SH3 domain of spectrin. Cells were serum-starved and plated on fibronectin-coated coverslips for 1 hour. Cells were fixed, permeabilized, and stained with antibody against HA epitope to detect transfected cells. Images are representative of four independent experiments. In all four experiments the transfection efficiency was 30%. (B) CHO cells expressing human IIbß3 integrin (control) or those that were also transiently transfected with HA-tagged SH3 domain, were serum-starved for 24 hours and plated on fibrinogen-coated dishes for 30 minutes, 1 hour or 3 hours. Cell lysates were incubated with GST-PBD beads, sedimented and bound Rac detected by western blotting. Lanes 1, 2 and 3 show bound Rac from lysates of cells expressing HA-tagged SH3 domain; lanes 4, 5 and 6 show bound Rac from control lysates. The bottom lanes show the amount of Rac in the total cell lysates. Data are representative of four independent experiments. Bar, 200 µm.

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