Dissecting focal adhesions in cells differentially expressing calreticulin: a microscopy study

Abstract

Background information: Our previous studies have shown that calreticulin, a Ca2+-binding chaperone located in the endoplasmic reticulum, affects cell-substratum adhesions via the induction of vinculin and N-cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected.

Results: The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference-reflection-microscopy-dark, vinculin- and paxillin-containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin-coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin-rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin-rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells.

Conclusions: We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.