An algorithm for detecting internalized, FM 1–43 stained membrane in epithelia demonstrates greater membrane internalization in low density cell clusters vs high density monolayers

Abstract

The membrane dye FM 1–43 rapidly stains a cell’s extracellular membrane including any membrane exocytosed, a property used to quantify exocytosis in neurons. In epithelia, fluctuations in basal and lateral intracellular space staining render FM 1–43 unreliable as a marker of exocytosis. However, membrane retrieved in the presence of FM 1–43 retains the dye, and washout of external stain reveals punctate, fluorescent particles. We developed an algorithm to quantify the retained fluorescent puncta in cells using images acquired with serial sectioning, and tested the algorithm on both coverslip-grown cell clusters and filter-grown intact monolayers. Serial sectioning was performed with epifluorescence microscopy, and a subset of experiments used the fixable variant of FM 1–43 to allow serial sectioning of the same preparation using both epifluorescence and confocal microscopy. Tests using an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that: 1) the algorithm identified the same number of internalized particles for a set condition with either epifluorescence or confocal microscopy; 2) low density clusters with an average of 5 cells exhibited significantly more internalized particles per cell (316 ± 31) than filter grown monolayers (9 ± 3) or high density clusters with an average of 120 cells (8 ± 3); and 3) ATP stimulation significantly increased the number of internalized particles in all preparations. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells. (CFF)