Cilia-cilia adhesions are glycoprotein-dependent, but not calcium- or disulfide- dependent. (A) Many adhesion proteins including integrins and cadherins require calcium. To assess the calcium requirement of the inter-cilia adhesion, cilia making contact were located, then the sample was moved to a solution of 2 mM EGTA, the same field of cells was re-located, and the cilia adhesion was monitored over time. The maximum intensity projections show that the adhesion between the cilia persisted long after the cadherin-based adhesions have been disrupted (cells round up) and adhesion of an additional cilium occurred in the absence of calcium (arrowhead in second panel). (B) Adhesion molecules containing immunoglobulin-like domains require disulfide bonds. We monitored cilia contact after addition of 10 mM DTT. The XZ projection image shows that the treatment causes the cells to deform and come off the filter, yet the cilia adhesion persists for more than 1 h. (C) We observed that leaving cells in the imaging conditions for 12 h resulted in an increase in the formation of contact relative to cells directly removed from the growth conditions (P value < 0.0005). We took advantage of this observation to assay the ability of the mannosidase II inhibitor, swainsonine, to prevent stable cilia contacts. We found that in the presence of 2 μg/mL swainsonine the number of cilia contacts was similar to cells directly removed from the growth media, indicating that mature glycoproteins promote the formation of cilia contacts (P < 0.005). Each value is calculated from more than 640 cilia in three different samples. (D) Side projections of cells in the imaging conditions for 12 h without (upper) or with (lower) swainsonine treatment. (E) During flagellar adhesion in Chlamydomonas reinhardtii the cilia localization of polycystin 2 increases. We used an antibody to polycystin 2 to demonstrate that polycystin 2 localization is similar in cilia that adhere to other cilia and cilia that are free. Scale bars are 5 μm.