Annexin V is a major intracellular calcium-binding protein in human foreskin fibroblasts. Immunocytochemistry revealed that annexin V was localized in the nucleus and throughout the cytoplasm in human foreskin fibroblasts. The presence of annexin V in the nucleus was variable depending on the growth state. Nuclear staining was strongest in proliferating cells immediately after sub-culture, and decreased on prolonged culture without changing the culture medium. The cytoplasmic location of annexin V was not greatly affected by the same conditions. Refeeding cells with fresh serum restored annexin V to the nuclei of all cells within in 24 h indicating that nuclear localization of annexin V is dependent on serum factors.

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and p1 values similar to those of annexins I, II, Ill, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by Kdepolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to nonphospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus. Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

Annexins are a family of proteins implicated in a number of cellular processes involving calcium. We studied annexins I, II, IV, V and VI and found that they are all present in human foreskin fibroblasts and, from immunocytochemical studies, have distinct locations in the cell. Only annexin IV and annexin V have unstructured cytoplasmic staining patterns consistent with predominantly cytosolic locations. Annexin VI partially colocalizes with the endoplasmic reticulum. In contrast, annexins I and II are both associated with the plasma membrane with annexin II having a very homogeneous staining compared with the punctate pattern observed for annexin I. Annexins I, IV and V are all present in the nucleus at higher concentrations than in the cytoplasm. Treatment of cells with the calcium ionophore A23187 to raise intracellular calcium, results in relocations of annexin II, IV, V and VI. Intranuclear annexins IV and V relocate to the nuclear membrane whereas the cytosolic pools of these annexins relocate to the plasma membrane. Annexin II relocates to granular structures at the plasma membrane whereas annexin VI relocates to a more homogeneous distribution on the plasma membrane. These results are consistent with an important role for annexins in mediating the calcium signal at the plasma membrane and within the nuclei of fibroblasts.