Bovine lactadherin as a calcium-independent imaging agent of phosphatidylserine expressed on the surface of apoptotic HeLa cells

Abstract

Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V.

Figure 1

Lactadherin staining of apoptotic cells at low magnification. Cells are stained with 5 nM Alexa Fluor 488–labeled bovine lactadherin and propidium iodide (PI). (A–C) HeLa cells induced for 3 hr to apoptosis by 10 μM staurosporine. The images represent selected slides of a confocal z-stack of the same area, with A located nearest to the glass surface. (D) Non-apoptotic HeLa cells stained with 5 nM of Alexa Fluor 488–labeled bovine lactadherin. The observed morphology of staurosporine-induced Hela cells is characterized by reduction of cell volume, ruffling, and vesiculation. In addition, a relatively large amount of thin, elongated filopodia-like areas appears. Little PI staining is evident, suggesting that few cells have entered secondary necrosis. Lactadherin stains avidly in the thin, elongated filopodia-like areas, as well as cell rims and vesicles. Bar = 20 μm.

Figure 2

Validation of lactadherin-specific binding at high magnification. HeLa cells are induced for 3 hr to apoptosis by 10 μM staurosporine and stained with fluorescently labeled lactadherin. Lactadherin staining is normalized to appear green. (A) Cells are stained with 10 nM Alexa Fluor 633–labeled lactadherin. (B) Cells are stained with 10 nM Alexa Fluor 488–labeled lactadherin and 200 nM goat antibody against the C2 domain of lactadherin. (C) Cells are stained with 10 nM Alexa Fluor 633 coincubated with 250× molar surplus of Ser-His-Arg-Gly-Asp-Val-Phe peptide. (D) Cells stained with CellBrite Green cytoplasmic membrane staining kit. The observed lactadherin staining is independent of Arg-Gly-Asp–mediated adhesion, whereas antibodies against the PS binding C2 domain result in clear staining inhibition. Furthermore, staining with the NeuroDIO membrane stain available in the Cellbrite Green cytoplasmic membrane staining kit confirms that the observed affinities of lactadherin staining can indeed be assigned to membrane-covered areas. Bar = 10 μm.

Figure 3

Lactadherin and annexin V costaining of apoptotic cells at high magnification. Cells are stained with 10 nM Alexa Fluor 633–labeled bovine lactadherin and 10 nM Alexa Fluor 488–labeled annexin V. Lactadherin is normalized to appear green, and annexin V is normalized to appear red. Areas with costaining appear yellow. (A–D) HeLa cells induced for 3 hr to apoptosis by 10 μM staurosporine. The images represent selected slides of a confocal z-stack of the same area, with A located nearest to the glass surface. There is a markedly different binding pattern of lactadherin and annexin V. Lactadherin mainly performs staining of the cellular rim and of blebbing vesicles (white arrowhead), as well as staining of thin filopodia-like cell appendages (black arrowhead). Annexin V stains mainly as bright spots (black arrow), consistent with internalized annexin V, or on blebbing membranes and the rim of cells. (E–G) Images represent one unmerged z-stack near the plane of cell attachment, with E being lactadherin staining, F being annexin V staining, and G showing the phase contrast image. (H) The merged image of E–G. Bar = 10 μm.