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

Abstract

The feasibility of the renal epithelial LLC-PK1 cell line as a model for cell 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 prepared. Fractured freeze-dried mitotic cells showed well-preserved organelles as revealed by fluorescence imaging of rhodamine-123 and C6-NBD-ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze-dried dividing cells revealed minimal surface topography that does not interfere in isotopic imaging of both positive (39K, 23Na, 24Mg, 40Ca, etc.) and negative (31P, 35Cl, etc.) secondaries with a CAMECA IMS-3f ion microscope. Mitotic cells revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of 39K+ and 23Na+) as revealed by K : Na ratios of approximately 10. Structurally damaged mitotic cells could be identified by their reduced K : Na ratios and an excessive loading of calcium. Quantitative three-dimensional SIMS analysis was required for studying subcellular calcium distribution in dividing cells. The LLC-PK1 model also allowed SIMS studies of M-phase arrested cells with mitosis-arresting drugs (taxol, monastrol and nocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.