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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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Time relations between morphological and membrane protein changes during spontaneous inside-out vesiculation of red cell membranes. Red cells were lysed and resuspended at 50 % equivalent haematocrit in L at 0 °C. The suspension was kept at 0 °C in an ice-bath. A 5-μL sample was placed between slide and coverslip on a temperature-controlled stage, initially set at 4 °C, of a Zeiss photomicroscope under Nomarski (×1,000) observation. Vesiculation was initiated (t = 0) both in the suspension and slide by transfer to a water bath at 37 °C and by rising the stage temperature to 37 °C, respectively. Suspension samples for membrane (left of paired columns) and supernatant (right of paired columns) proteins were taken at the indicated times and processed as described in “Methods”. Nomarski photo-images were taken at the indicated times and are shown in the figure lined up above the time-corresponding membrane protein columns. Note that the reduction of spectrin from pellets (bands 1 and 2, Fig 3) and the appearance of spectrin in supernatants become detectable at about 5 min, just before large-scale vesicular sealing as indicated by haemoglobin trapping (bottom band). Allowing for imperfect synchronization, the results suggest large-scale spectrin retention during the early dynamic stages of the spontaneous vesiculation process
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Fig4: Time relations between morphological and membrane protein changes during spontaneous inside-out vesiculation of red cell membranes. Red cells were lysed and resuspended at 50 % equivalent haematocrit in L at 0 °C. The suspension was kept at 0 °C in an ice-bath. A 5-μL sample was placed between slide and coverslip on a temperature-controlled stage, initially set at 4 °C, of a Zeiss photomicroscope under Nomarski (×1,000) observation. Vesiculation was initiated (t = 0) both in the suspension and slide by transfer to a water bath at 37 °C and by rising the stage temperature to 37 °C, respectively. Suspension samples for membrane (left of paired columns) and supernatant (right of paired columns) proteins were taken at the indicated times and processed as described in “Methods”. Nomarski photo-images were taken at the indicated times and are shown in the figure lined up above the time-corresponding membrane protein columns. Note that the reduction of spectrin from pellets (bands 1 and 2, Fig 3) and the appearance of spectrin in supernatants become detectable at about 5 min, just before large-scale vesicular sealing as indicated by haemoglobin trapping (bottom band). Allowing for imperfect synchronization, the results suggest large-scale spectrin retention during the early dynamic stages of the spontaneous vesiculation process

Mentions: The time-dependent changes in RBC membrane proteins throughout the spontaneous IO vesiculation process were followed in parallel with morphological changes under Nomarski optics observation (Fig. 4). During the first 5 min, we see profound morphological changes along previously described patterns [25] with hardly any detectable change in membrane proteins. The release of spectrin and actin to the supernatant becomes detectable only after the 5-min sample in this series. Spectrin detachment from the membranes to the supernatant is progressively evident in the 6- and 8-min samples, and the weaker actin band is clearly detectable in the 8-min supernatant sample. Between the fifth and sixth minute, there is an abrupt increase in the membrane-associated monomeric Hb band, in parallel with late formation of vesicles. These results clearly show that the main cytoskeletal mesh components, spectrin and actin, remain associated with the cell membrane during the dynamic morphological changes leading to vesiculation and that they only become fully dissociated at the final vesicle sealing stage, when Hb becomes trapped within sealed vesicles.Fig. 4


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

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

Time relations between morphological and membrane protein changes during spontaneous inside-out vesiculation of red cell membranes. Red cells were lysed and resuspended at 50 % equivalent haematocrit in L at 0 °C. The suspension was kept at 0 °C in an ice-bath. A 5-μL sample was placed between slide and coverslip on a temperature-controlled stage, initially set at 4 °C, of a Zeiss photomicroscope under Nomarski (×1,000) observation. Vesiculation was initiated (t = 0) both in the suspension and slide by transfer to a water bath at 37 °C and by rising the stage temperature to 37 °C, respectively. Suspension samples for membrane (left of paired columns) and supernatant (right of paired columns) proteins were taken at the indicated times and processed as described in “Methods”. Nomarski photo-images were taken at the indicated times and are shown in the figure lined up above the time-corresponding membrane protein columns. Note that the reduction of spectrin from pellets (bands 1 and 2, Fig 3) and the appearance of spectrin in supernatants become detectable at about 5 min, just before large-scale vesicular sealing as indicated by haemoglobin trapping (bottom band). Allowing for imperfect synchronization, the results suggest large-scale spectrin retention during the early dynamic stages of the spontaneous vesiculation process
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Time relations between morphological and membrane protein changes during spontaneous inside-out vesiculation of red cell membranes. Red cells were lysed and resuspended at 50 % equivalent haematocrit in L at 0 °C. The suspension was kept at 0 °C in an ice-bath. A 5-μL sample was placed between slide and coverslip on a temperature-controlled stage, initially set at 4 °C, of a Zeiss photomicroscope under Nomarski (×1,000) observation. Vesiculation was initiated (t = 0) both in the suspension and slide by transfer to a water bath at 37 °C and by rising the stage temperature to 37 °C, respectively. Suspension samples for membrane (left of paired columns) and supernatant (right of paired columns) proteins were taken at the indicated times and processed as described in “Methods”. Nomarski photo-images were taken at the indicated times and are shown in the figure lined up above the time-corresponding membrane protein columns. Note that the reduction of spectrin from pellets (bands 1 and 2, Fig 3) and the appearance of spectrin in supernatants become detectable at about 5 min, just before large-scale vesicular sealing as indicated by haemoglobin trapping (bottom band). Allowing for imperfect synchronization, the results suggest large-scale spectrin retention during the early dynamic stages of the spontaneous vesiculation process
Mentions: The time-dependent changes in RBC membrane proteins throughout the spontaneous IO vesiculation process were followed in parallel with morphological changes under Nomarski optics observation (Fig. 4). During the first 5 min, we see profound morphological changes along previously described patterns [25] with hardly any detectable change in membrane proteins. The release of spectrin and actin to the supernatant becomes detectable only after the 5-min sample in this series. Spectrin detachment from the membranes to the supernatant is progressively evident in the 6- and 8-min samples, and the weaker actin band is clearly detectable in the 8-min supernatant sample. Between the fifth and sixth minute, there is an abrupt increase in the membrane-associated monomeric Hb band, in parallel with late formation of vesicles. These results clearly show that the main cytoskeletal mesh components, spectrin and actin, remain associated with the cell membrane during the dynamic morphological changes leading to vesiculation and that they only become fully dissociated at the final vesicle sealing stage, when Hb becomes trapped within sealed vesicles.Fig. 4

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