Cell Cycle-Dependent Localization of Voltage-Dependent Calcium Channels and the Mitotic Apparatus in a Neuroendocrine Cell Line(AtT-20)

Figure 1

Calcium channel CaV1.2 and CaV1.3 localization in mitotic cells. 1(A) compares CaV1.2 staining in AtT-20 cells (a)–(c), PC-12 cells (d), and INS-1 cells (e)–(g). CaV1.2 is located with kinetechores at the “poleward side” during metaphase and anaphase ((a), (b): inset) in the AtT-20 cells. Midbody CaV1.2 staining is observed in both AtT-20 cells and PC-12 cells (c), (d) but not in INS-1 cells (g). 1(B) is a similar comparison for CaV1.3 and shows staining at the mid-spindle zone only in AtT-20s during telophase (a). In contrast, all three neuroendocrine cell types display CaV1.3 staining at the midbody during cytokinesis ((B): (b), (c), and (e)). 1(C) shows control staining of Cos 7 cells; in particular, note that there is no nonspecific staining at the midbody of these cells (1(C) (c), (e)). CaV1.2 and CaV1.3 are stained with antibodies specific to alpha subunit sequences for CaV1.2 and CaV1.3 (see Methods, Alamone Labs) and detected with secondary goat anti-rabbit antibody Alexa 555 (red); Microtubules are stained with DM1A and goat anti-mouse Alexa 647 (blue); kinetechores are stained with anti-CREST antibody and Alexa 488 (green). Bleedthrough between microtubule and CREST staining channels was corrected for as described in methods.