Calcium metabolism is altered in the absence of syndecan-4. (A and B) FRET analysis using Twitch-1 reveals elevated calcium levels in s4ko MEF compared with matching wt counterparts. Re-expression of Sdc4 in s4ko cells reduces calcium to wt levels. n = 115 for wtMEF; n = 90 for s4ko MEF; n = 120 for s4ko + wt MEF. (C and D) Reduction of syndecan-4 in rat embryonic fibroblasts leads to elevated cytosolic calcium. n = 63 in D. (E–G) Ectopic expression of Sdc4 mutant forms lacking heparan sulfate chains (Sdc4-AAA) or a truncated cytoplasmic domain (Sdc-Δ191I) do not restore calcium to wt levels. However, expression of Sdc4 mutant lacking the C2 domain (Sdc-Δ199E) resulted in reduced calcium levels. n = 55–120 per condition. (H) Diagram of fibronectin domains and interactions of syndecan-4 leading to PKCα activation. (I–K) wt MEFs plated on FnIII_6–10 protein have elevated cytosolic calcium levels compared with those plated on full-length fibronectin or serum-coated surfaces, but undergo reduction in response to FnIII_12–15 protein addition (I). (J) s4ko MEF on FnIII_6–10 protein or full-length fibronectin possessed similar, high calcium levels that are unchanged by addition of FnIII_12–15 protein. (K) Treating wt MEFs plated on FnIII_6–10 with GF109203x did not increase calcium levels but in this case the addition of FnIII_12–15 failed to reduce the cytosolic calcium level. Additionally, wt MEF on whole fibronectin elevate calcium in response to PKC inhibition that is also unchanged after FnIII_12–15 addition. See also Videos 1, 2, 3, and 4. Data are the mean ± SEM. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; unpaired t test.