Calcium Signalling Joint Meeting with the Belgian Society for Cell Biology and The European Calcium Society

AN INVESTIGATION INTO THE PHYSIOLOGICAL ROLE OFANNEXIN A11 A. Tomas and S.E. MossDivision of Cell Biology, Institute of Ophthalmology, University College London, London (UK) Annexin A11 is a calcium-dependent phospholipid-binding protein that interacts in vitro with the EF-hand protein, Calcyclin. It is broadly distributed and has the longestN-terminal domain (197 aa) of all known annexins, which contains the binding sitefor Calcyclin. This unique N-terminal domain is alternatively spliced generating two different isoforms that differ in their capacity to bind to Calcyclin. Since Calcyclin isoverexpressed in tumor cells with the mRNA levels specifically increased in the G1phase of the cell cycle, it is possible that the Annexin A11-Calcyclin complex may play a role in cell proliferation and division. Annexin A11 is also known to be a targetfor cellular Ser/Thr and Tyr-kinases, suggesting that phosphorylation could regulateAnnexin A11 function in living cells. Annexin A11 may also be involved in calcium-regulated secretory processes, as it has been localised to various types of neutrophil-specific and insulin granules, and antibodies against Annexin A11 inhibitcalcium-dependent insulin secretion. We employed a number of different approaches to provide insights into the physiological function of Annexin A11. Using immunofluorescence techniques, weinvestigated the subcellular localisation of Annexin A11 in the human cell line A431.We followed Annexin A11 localisation throughout the cell cycle, and its relocalisation in response to changes in the intracellular calcium levels. We studied the levels oftyrosine phosphorylation of Annexin A11 in response to several agonists, and arecurrently deriving an annexin A11 knock-out in the chicken DT40 cell line. We arealso overexpressing either the aminoor the carboxy-terminal domain of human Annexin A11 to assess any potential dominant negative effects in vivo. THE ROLE OF ANNEXIN A5 IN APOPTOSISB. P. Young, T.E. Hawkins and S.E. Moss,Division of Cell Biology, Institute of Ophthalmology, 11-43 Bath Street, London (UK) The annexins are a family of ubiquitously expressed Ca-dependent phospholipid bindingproteins which have been implicated in a variety of cellular processes, including vesicletrafficking and Ca channel activity. To help improve our understanding of the physiologicalroles of the annexins, we have knocked out the annexin A5 gene in the DT40 chicken pre-Bcell line. Anx5 cells are viable, yet display several phenotypes, the most striking of which isresistance to drug-induced, Ca-dependent apoptosis. In contrast, Anx5 cells are stillsusceptible to apoptosis induced by irradiation with UVA/B light. To further understand the role of annexin A5 in apoptosis, a number of strategies are beingemployed. The degree to which Anx5 cells are resistant to apoptosis is being analysed bythorough kinetic experiments, which will determine whether Annexin A5 merely delays theonset of apoptosis or completely inhibits it. Using recently developed direct protein deliverytechniques, recombinant Annexin A5 can be introduced into cells at different levels in bothwild type and knockout cells, and the subsequent effects on apoptosis susceptibilitymonitored. One possibility is that knocking out annexin A5 may have altered the expression of othergenes involved in the apoptotic process. To investigate this, the levels of known apoptoticproteins are being analysed at the protein and mRNA level, as well as a more thoroughanalysis of gene expression using subtractive PCR methods. Finally, live cell imaging usingGFP-tagged Annexin A5 is being utilised to investigate whether Annexin A5 relocalises inresponse to apoptotic stimuli.