Fluorescent Probe for Transmembrane Dynamics during Osmotic Effects

Abstract

Membrane tension pores determine organelle dynamics and functions, giving rise to physical observables during the cell death process. While fluorescent organelle-targeted probes for specific chemical analytes are increasingly available, subcellular dynamic processes involving not only chemical parameters but also physicochemical and physical parameters are uncommon. Here, we report a mitochondrial chemical probe, named RCN, rationally designed to monitor osmotic effects during transmembrane tension pore formation by using local mitochondrial polarity and a subcellular localization redistribution property of the probe. Utilizing fluorescence spectroscopy, high-resolution confocal imaging, and spectrally resolved confocal microscopy, we provide a new correlation between mitochondrial dynamics and bleb vesicle formation using osmotic pressure stimuli in the cell, where the mitochondrial local polarity was found to drastically increase. The RCN provides a reliable protocol to assess transmembrane pore formation driven by osmotic pressure increments through local polarity variations and is a more robust physicochemical parameter allowing the health and decease status of the cell to be measured.

Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress

“…It is also in line with a recent report on a solvatochromic mitochondria-specific probe. 54 The peculiar behavior of lysosomes (e.g. lower polarity and higher LOP) is probably because they are expected to shrink, which induces higher lipid ordering at the inner leaflet of the vesicles, where NRLyso locates.…”

“…34,[45][46]49 It revealed that the intracellular biomembranes are significantly more polar (less ordered) than plasma membranes. 46 Second approach exploited specific targeting of solvatochromic dyes to organelle of interest, 5,25,[35][36][50][51][52][53] including mitochondria 32,50,54 and ER. 35 However, these studies were focused on targeting one particular organelle.…”

“…The first one used a non-specific lipophilic probe that redistributes in biomembranes of different organelles within the cell, thus allowing for polarity and lipid order mapping irrespectively to a given organelle. ,,, It revealed that the intracellular biomembranes are significantly more polar (less ordered) than plasma membranes . The second approach exploited the specific targeting of solvatochromic dyes to the organelle of interest, ,,,,− including mitochondria ,, and ER . However, these studies were focused on targeting one particular organelle.…”

Tuning the Cell Uptake and Subcellular Distribution in BODIPY–Carboranyl Dyads: An Experimental and Theoretical Study

“…It is worth mentioning that BODIPY‐Ref also exhibited interesting membrane permeability with a non‐specific localization in the perinuclear zone. Further, this fluorophore was able to specifically localize apoptotic vesicles and cellular blebs during cell oxidative stress [25] . The low‐dose stimulus with H 2 O 2 was used in order for the blebs to be well‐contrasted under the green channel illumination, allowing a bright in‐plane bleb visualization with clean green‐emission on these membranes.…”

“…Further, this fluorophore was able to specifically localize apoptotic vesicles and cellular blebs during cell oxidative stress. [25] The low-doses timulus with H 2 O 2 wasu sed in order for the blebst ob ew ell-contrasted under the green channel illumination, allowing ab right in-plane bleb visualization with cleangreen-emission on these membranes. At this point, we were motivated to investigate the origin of the divergentc ell uptake efficiency and subcellular localization of these molecules, with the most striking differencesa rising from BODIPY-o-Ph-CB and BODIPY-m-Ph-CB.…”