Abstract Title

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Abstract

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl-D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

Modified Abstract

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. It is accompanied by an almost 10% volume increase. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive Na channels. Low-sodium choline-, but not N-methyl-D-glucamine-based, medium, also supports swelling; this indicates that the membrane becomes permeable to choline as well. By using fluorescent dyes, we have shown that cell membranes during the blebbing phase become selectively permeable to substrates of organic cation transporters. Our results demonstrate a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

The research was partially supported by the University Research Council grant to Michael Model.

Research Category

Biomedical Sciences

Primary Author's Major

Biology

Mentor #1 Information

Dr.Michael Model

Presentation Format

Poster

Start Date

March 2016

Research Area

Cell Biology

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Mar 15th, 1:00 PM

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl-D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.