Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA116

Poster Communications

The Na+/HCO3- cotransporter NBCn1 in cell cycle regulation - possible role for an unexpected localization?

M. Severin1, D. Christensen1, J. Vogensen1, M. Flinck1, S. Kramer1, E. Sørensen1, J. Schnipper1, I. Axholm1, S. F. Pedersen1

1. Faculty of biology, University of Copenhagen, Copenhagen, Denmark.

The net acid extruding Na+/HCO3- cotransporter NBCn1 (Slc4a7) is widely expressed in the plasma membrane of mammalian tissues. Work from us and others has implicated NBCn1 in breast tumor development and NBCn1 knockdown or -knockout reduces tumor growth and proliferation in vivo (1;2). We recently demonstrated that stable knockdown of NBCn1 in MCF-7 breast cancer cells leads to slower cell cycle progression in synchronized cells, in particular in G2/M transition (3). Accordingly, doubling time was increased and Ki-67 staining decreased in asynchronous MCF-7 cells. Cell cycle regulatory proteins were affected in congruence with this pattern, but no mechanism could be identified (3). The aim of our ongoing work is to identify mechanisms involved in NBCn1-mediated cell cycle regulation. We performed a GST pulldown of the C-terminal of NBCn1 in MCF-7 cell lysates. Surprisingly, strong hits included End-binding protein (EB)1, associated with the mitotic spindle, and three centrosomal/ciliary base-located proteins, CEP135, CEP164, and CEP290. Co-immunoprecipitation of NBCn1 with CEP135 as well as the reverse pulldown confirmed this interaction. These data show that at least in cell lysates, NBCn1 interacts with centrosome- and spindle proteins. Fluorescence imaging using native and exogenously expressed NBCn1, and three different antibodies, as well as live imaging of GFP-tagged NBCn1, indicates localization to spindle, midbodies, and centrosome in a variety of cancer and non-cancer cells from several species (MCF-7, HeLa B, Panc-1, NIH-3T3, MDCK II, RPE-1). Ongoing work using a series of tagged and truncated NBCn1 variants is addressing the specificity of this pattern for NBCn1. If interaction can be validated in intact cells, we will test the hypothesis that NBCn1 may play a role in the regulation of pH in the spindle/centrosome region. This is in congruence with the known pH sensitivity of mitotic spindle formation, and with clear evidence from others that during mitosis a number of membrane proteins localize to the spindle and/or midbodies. Some membrane proteins appear to have secondary roles related to cytokinesis, but the role of most membrane proteins at the spindle proteome is unknown.

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