Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy: LLC-PK1 epithelial cells as a model forsubcellular isotopic imaging

The feasibility of the renal epithelial LLC-PK1 cell line as a model for ce ll division studies with secondary ion mass spectrometry (SIMS) was tested. In this cell line, cells undergoing all stages of mitosis and cytokinesis remained firmly attached to the substrate and could be cryogenically prepar ed. Fractured freeze-dried mitotic cells showed well-preserved organelles a s revealed by fluorescence imaging of rhodamine-123 and C-6-NBD-ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze-dried dividing cells revealed minimal surface topograp hy that does not interfere in isotopic imaging of both positive (K-39, Na-2 3, Mg-24, Ca-40, etc.) and negative (P-31, Cl-35, etc.) secondaries with a CAMECA IMS-3f ion microscope. Mitotic cells revealed well-preserved intrace llular ionic composition of even the most diffusible ions (total concentrat ions of K-39(-/-) and Na-23(+)) as revealed by K : Na ratios of approximate ly 10. Structurally damaged mitotic cells could be identified by their redu ced K : Na ratios and an excessive loading of calcium. Quantitative three-d imensional SIMS analysis was required for studying subcellular calcium dist ribution in dividing cells. The LLC-PK1 model also allowed SIMS studies of M-phase arrested cells with mitosis-arresting drugs (taxol, monastrol and n ocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.