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Dynamic morphology and cytoskeletal protein changes during spontaneous inside-out vesiculation of red blood cell membranes.

Tiffert T, Lew VL - Pflugers Arch. (2014)

Bottom Line: We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh.These results support the proposed role of spectrin-actin in spontaneous vesiculation.The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK, jtt1000@cam.ac.uk.

ABSTRACT
Vesicle preparations from cell plasma membranes, red blood cells in particular, are extensively used in transport and enzymic studies and in the fields of drug delivery and drug-transport interactions. Here we investigated the role of spectrin-actin, the main components of the red cell cortical cytoskeleton, in a particular mechanism of vesicle generation found to be relevant to the egress process of Plasmodium falciparum merozoites from infected red blood cells. Plasma membranes from red blood cells lysed in ice-cold media of low ionic strength and free of divalent cations spontaneously and rapidly vesiculate upon incubation at 37 °C rendering high yields of inside-out vesicles. We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh. We showed that cytoskeletal-free membranes behave like a two-dimensional fluid lacking shape control, that spectrin-actin remain attached to vesiculating membranes for as long as spontaneous movement persists, that most of the spectrin-actin detachment occurs terminally at the time of vesicle sealing and that naked membrane patches increasingly appear during vesiculation. These results support the proposed role of spectrin-actin in spontaneous vesiculation. The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

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Effect of magnesium ions on the time-dependent patterns of membrane protein changes during spontaneous vesiculation. The experimental protocol was similar to that described in Fig 4. Samples for membrane and supernatant proteins shown on the left panels were taken before (t = 0) and after switching the temperature of the ghost suspension from 0 to 37 °C, at the times indicated under each column (in min). Left panels show the patterns obtained from Mg-inactivated samples immediately processed for SDS–gel electrophoresis, as reported in “Methods” (Mg inactivation). Right panels show the patterns recovered from duplicate Mg-inactivated samples from each of the originally timed samples after 1 h of incubation at 37 °C before processing for SDS–gel electrophoresis (Mg inactivation/incubation). Extreme left and right columns in each of the four panels are molecular weight standards. Top panels show membrane proteins; corresponding bottom panels show bands of proteins lost to the supernatant. Notwithstanding imperfect synchronization, it is clear that there is no large-scale spectrin loss (bands 1 and 2, see Fig 3) from membranes to supernatants during the first 4 to 5 min, the most dynamic stages of the spontaneous vesiculation process. Large-scale spectrin loss occurs concurrently with haemoglobin retention in the membrane protein gels (6-min sample) reflecting vesicular sealing. The similitude of the two membrane protein gels (top panels), particularly for the samples taken during the first 4 to 5 min of incubation, supports the view that vesiculation arrest by Mg2+ results from prevention of spectrin detachment, probably through spectrin cross-linking
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Fig5: Effect of magnesium ions on the time-dependent patterns of membrane protein changes during spontaneous vesiculation. The experimental protocol was similar to that described in Fig 4. Samples for membrane and supernatant proteins shown on the left panels were taken before (t = 0) and after switching the temperature of the ghost suspension from 0 to 37 °C, at the times indicated under each column (in min). Left panels show the patterns obtained from Mg-inactivated samples immediately processed for SDS–gel electrophoresis, as reported in “Methods” (Mg inactivation). Right panels show the patterns recovered from duplicate Mg-inactivated samples from each of the originally timed samples after 1 h of incubation at 37 °C before processing for SDS–gel electrophoresis (Mg inactivation/incubation). Extreme left and right columns in each of the four panels are molecular weight standards. Top panels show membrane proteins; corresponding bottom panels show bands of proteins lost to the supernatant. Notwithstanding imperfect synchronization, it is clear that there is no large-scale spectrin loss (bands 1 and 2, see Fig 3) from membranes to supernatants during the first 4 to 5 min, the most dynamic stages of the spontaneous vesiculation process. Large-scale spectrin loss occurs concurrently with haemoglobin retention in the membrane protein gels (6-min sample) reflecting vesicular sealing. The similitude of the two membrane protein gels (top panels), particularly for the samples taken during the first 4 to 5 min of incubation, supports the view that vesiculation arrest by Mg2+ results from prevention of spectrin detachment, probably through spectrin cross-linking

Mentions: The experimental protocol for the spontaneous formation of IOVs is illustrated in Fig. 1. Briefly, fresh venous blood was obtained from healthy donors after informed consent, using heparinized syringes. Red blood cells were washed three times with 10 vol of a solution containing (in mM) NaCl 142, KCl 3, HEPES-Na (pH 7.5) 10 and EGTA or EDTA 0.1, to chelate extracellular divalent cations. Residual plasma, buffy coat and topmost cell layer were removed after each wash. For vesiculation, the washed red cells were lysed in 50–100 vol of ice-cold solution “L” (in mM): HEPES-Na (pH 7.5) 2.5 and EGTA 0.1. The lysed cells were immediately spun at 15,000×g for 15–20 min at 0–5 °C, forming a pink pellet on top of a tiny dark button at the bottom of the centrifuged tube. The supernatant was discarded, and the pink ghost pellet was gently transferred to a new tube avoiding any contact and mixing with the dark adherent button at the bottom of the tube. This button contains residual blood cell contaminants rich in proteases capable of drastically altering the electrophoretic patterns studied here if retained, or imperfectly removed [43]. Conservation of the sodium dodecyl sulphate (SDS)–gel electrophoretic pattern of red cell membrane proteins in the current controls, even after prolonged incubations (see Fig. 5), documents the effectiveness of contaminant protease removal. The transferred ghost pellet was resuspended in ice-cold solution L at an equivalent hematocrit (relative to the original volume of cells) of 50–100 % and the suspension pre-incubated in the ice-bath for 30–60 min to optimize synchronized vesiculation in the ghost population when subsequently incubated at 37 °C. Vesiculation was initiated by transferring the suspension to a water bath at 37 °C. Duplicate 0.1-ml samples of the suspension were taken before and after this transfer at the time intervals indicated in the figures and diluted ten-fold into microfuge tubes containing 0.9 mL of solution L with 0.1–0.5 mM MgCl2 to halt further vesiculation [11]. Phase contrast and Nomarski observations and photomicroscopy (Zeiss Photomicroscope III) were performed on these unfixed samples using ×l00 oil immersion objective lenses and temperature-controlled slides.Fig. 1


Dynamic morphology and cytoskeletal protein changes during spontaneous inside-out vesiculation of red blood cell membranes.

Tiffert T, Lew VL - Pflugers Arch. (2014)

Effect of magnesium ions on the time-dependent patterns of membrane protein changes during spontaneous vesiculation. The experimental protocol was similar to that described in Fig 4. Samples for membrane and supernatant proteins shown on the left panels were taken before (t = 0) and after switching the temperature of the ghost suspension from 0 to 37 °C, at the times indicated under each column (in min). Left panels show the patterns obtained from Mg-inactivated samples immediately processed for SDS–gel electrophoresis, as reported in “Methods” (Mg inactivation). Right panels show the patterns recovered from duplicate Mg-inactivated samples from each of the originally timed samples after 1 h of incubation at 37 °C before processing for SDS–gel electrophoresis (Mg inactivation/incubation). Extreme left and right columns in each of the four panels are molecular weight standards. Top panels show membrane proteins; corresponding bottom panels show bands of proteins lost to the supernatant. Notwithstanding imperfect synchronization, it is clear that there is no large-scale spectrin loss (bands 1 and 2, see Fig 3) from membranes to supernatants during the first 4 to 5 min, the most dynamic stages of the spontaneous vesiculation process. Large-scale spectrin loss occurs concurrently with haemoglobin retention in the membrane protein gels (6-min sample) reflecting vesicular sealing. The similitude of the two membrane protein gels (top panels), particularly for the samples taken during the first 4 to 5 min of incubation, supports the view that vesiculation arrest by Mg2+ results from prevention of spectrin detachment, probably through spectrin cross-linking
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4233320&req=5

Fig5: Effect of magnesium ions on the time-dependent patterns of membrane protein changes during spontaneous vesiculation. The experimental protocol was similar to that described in Fig 4. Samples for membrane and supernatant proteins shown on the left panels were taken before (t = 0) and after switching the temperature of the ghost suspension from 0 to 37 °C, at the times indicated under each column (in min). Left panels show the patterns obtained from Mg-inactivated samples immediately processed for SDS–gel electrophoresis, as reported in “Methods” (Mg inactivation). Right panels show the patterns recovered from duplicate Mg-inactivated samples from each of the originally timed samples after 1 h of incubation at 37 °C before processing for SDS–gel electrophoresis (Mg inactivation/incubation). Extreme left and right columns in each of the four panels are molecular weight standards. Top panels show membrane proteins; corresponding bottom panels show bands of proteins lost to the supernatant. Notwithstanding imperfect synchronization, it is clear that there is no large-scale spectrin loss (bands 1 and 2, see Fig 3) from membranes to supernatants during the first 4 to 5 min, the most dynamic stages of the spontaneous vesiculation process. Large-scale spectrin loss occurs concurrently with haemoglobin retention in the membrane protein gels (6-min sample) reflecting vesicular sealing. The similitude of the two membrane protein gels (top panels), particularly for the samples taken during the first 4 to 5 min of incubation, supports the view that vesiculation arrest by Mg2+ results from prevention of spectrin detachment, probably through spectrin cross-linking
Mentions: The experimental protocol for the spontaneous formation of IOVs is illustrated in Fig. 1. Briefly, fresh venous blood was obtained from healthy donors after informed consent, using heparinized syringes. Red blood cells were washed three times with 10 vol of a solution containing (in mM) NaCl 142, KCl 3, HEPES-Na (pH 7.5) 10 and EGTA or EDTA 0.1, to chelate extracellular divalent cations. Residual plasma, buffy coat and topmost cell layer were removed after each wash. For vesiculation, the washed red cells were lysed in 50–100 vol of ice-cold solution “L” (in mM): HEPES-Na (pH 7.5) 2.5 and EGTA 0.1. The lysed cells were immediately spun at 15,000×g for 15–20 min at 0–5 °C, forming a pink pellet on top of a tiny dark button at the bottom of the centrifuged tube. The supernatant was discarded, and the pink ghost pellet was gently transferred to a new tube avoiding any contact and mixing with the dark adherent button at the bottom of the tube. This button contains residual blood cell contaminants rich in proteases capable of drastically altering the electrophoretic patterns studied here if retained, or imperfectly removed [43]. Conservation of the sodium dodecyl sulphate (SDS)–gel electrophoretic pattern of red cell membrane proteins in the current controls, even after prolonged incubations (see Fig. 5), documents the effectiveness of contaminant protease removal. The transferred ghost pellet was resuspended in ice-cold solution L at an equivalent hematocrit (relative to the original volume of cells) of 50–100 % and the suspension pre-incubated in the ice-bath for 30–60 min to optimize synchronized vesiculation in the ghost population when subsequently incubated at 37 °C. Vesiculation was initiated by transferring the suspension to a water bath at 37 °C. Duplicate 0.1-ml samples of the suspension were taken before and after this transfer at the time intervals indicated in the figures and diluted ten-fold into microfuge tubes containing 0.9 mL of solution L with 0.1–0.5 mM MgCl2 to halt further vesiculation [11]. Phase contrast and Nomarski observations and photomicroscopy (Zeiss Photomicroscope III) were performed on these unfixed samples using ×l00 oil immersion objective lenses and temperature-controlled slides.Fig. 1

Bottom Line: We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh.These results support the proposed role of spectrin-actin in spontaneous vesiculation.The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

View Article: PubMed Central - PubMed

Affiliation: Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK, jtt1000@cam.ac.uk.

ABSTRACT
Vesicle preparations from cell plasma membranes, red blood cells in particular, are extensively used in transport and enzymic studies and in the fields of drug delivery and drug-transport interactions. Here we investigated the role of spectrin-actin, the main components of the red cell cortical cytoskeleton, in a particular mechanism of vesicle generation found to be relevant to the egress process of Plasmodium falciparum merozoites from infected red blood cells. Plasma membranes from red blood cells lysed in ice-cold media of low ionic strength and free of divalent cations spontaneously and rapidly vesiculate upon incubation at 37 °C rendering high yields of inside-out vesicles. We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh. We showed that cytoskeletal-free membranes behave like a two-dimensional fluid lacking shape control, that spectrin-actin remain attached to vesiculating membranes for as long as spontaneous movement persists, that most of the spectrin-actin detachment occurs terminally at the time of vesicle sealing and that naked membrane patches increasingly appear during vesiculation. These results support the proposed role of spectrin-actin in spontaneous vesiculation. The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

Show MeSH
Related in: MedlinePlus