Measuring Intracellular Viscosity in Conditions of Hypergravity

Abstract

Gravity-sensitive cellular responses are regularly observed in both specialized and nonspecialized cells. One potential mechanism for this sensitivity is a changing viscosity of the intracellular organelles. Here, we report a novel, to our knowledge, viscosity-sensitive molecular rotor based on mesosubstituted boron-dipyrrin used to investigate the response of viscosity of cellular membranes to hypergravity conditions created at the large diameter centrifuge at the European Space Agency Technology Centre. Mouse osteoblastic (MC3T3-E1) and endothelial (human umbilical vein endothelial cell) cell lines were tested, and an increase in viscosity was found with increasing hypergravity loading. This response is thought to be primarily biologically driven, with the potential for a small, instantaneous physical mechanism also contributing to the observed effect. This work provides the first, to our knowledge, quantitative data for cellular viscosity changes under hypergravity, up to 15 × g.

Copyright © 2019. Published by Elsevier Inc.

Figures

Figure 1

The molecular rotors 1 and 2. (a) The molecular structure of rotor 1 and rotor 2 is shown. (b) Fluorescence emission spectra of 2 in methanol/glycerol mixtures of varying concentrations with viscosities ranging from 0.6 to 1457.6 cP, excited at 450 nm, are shown. (c) A fluorescence intensity image showing representative staining of MC3T3-E1 cells with 2 at 1 × g (200-ms exposure), is shown. Scale bars, 10 μm. To see this figure in color, go online.

Figure 2

Fluorescence intensity images of MC3T3-E1 cells showing change in intensity of molecular rotor 2 at 1 × g (0 min), 15 × g (15 min), 10 × g (50 min), and 1 × g (80 min) using a 15-min ramp (200-ms exposure). Scale bars, 10 μm.

Figure 3

(a) Fluorescence response of 2 in MC3T3-E1 cells (black) as g level (green) is changed, with a 15-min ramp. (b) Control MC3T3-E1 cells showing no change in 2 fluorescence (black) as g level (green) is held at 1 × g are shown. (c) The fluorescence response of 2 in MC3T3-E1 cells (black) as g level (green) is changed with a 7.5-min ramp is shown. (d) A normalized fluorescence response of cells treated by previous hypergravity exposure and relaxation, before a second hypergravity exposure (black), compared with cells with no previous hypergravity exposure (blue) is shown. To see this figure in color, go online.

Figure 4

(a) Fluorescence response of 2 in HUVECs (black) as g level (green) is changed, with a 15-min ramp. (b) Fluorescence images of HUVECs showing change in intensity of 2 at 1 × g (t = 0 min) and 15 × g (t = 45 min) during a 15-min ramp (200-ms exposure), are shown. (c) The fluorescence response of 2 in HUVECs (black) as g level (green) is changed with a 7.5-min ramp is shown. (d) Control HUVECs showing an increase in 2 fluorescence intensity (black) as g level (green) is held at 1 × g are shown. (e) Fluorescence images of HUVECs showing change in intensity of 2 at 1 × g at various time points are shown. Scale bars, 10 μm. To see this figure in color, go online.