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8/13/2019 An actin barrier for resealing
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12.13.13
onThKim Dung (D1)
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Cell membrane resealing and current hypotheses
Phospholipid reorientation to reduce net free energy
Plasma membrane
Resealing small disruption
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Ca 2+triggers vesicle-vesicle fusion
Formation of a large patch vesicle
Vesicle patch hypothesis
Vesicle population increase
Resealing large disruption >1m
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Proposed role of actin depolymerization in resealing
No wound time (t=0) 1.
2. 3.
Actin Filaments Vesicle Actin depolymerization and vesicle fusion
Resealing by patch vesicle formation
(time 1 min)
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Purposes of this research:
- To clarify the role of actin filament
- To examine actin polymerization states during resealing- To assess the effect of actin filament depolymerization
agents on resealing
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Examination of large vesicle patch formation
Method of plasma membrane disruption:
-scratching under the presence of horseradish peroxidase (HRP)
(fixed within 15)
Cell culture:
-The rat gastric mucosal epithelial cell line, RGM1
Method of observation:
- Using transmission electron microscope to image regions of
cell cytoplasm bordering
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Large patch vesicles formation
TEM images of the cortex of
unwounded RGM1 cell
and wounded RGM1 cell
staining with HRP
Vesicle population increasing
Membrane disruption site
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Method of visualizing actin depolymerization
induced by disruption
Materials: Fixable fluorescenein-labelled dextran (FDx) using for
identify the bordering on scratch
Tetramethylrodamin isothiolcynate (TRITC)-phalloidin
using for identifying distribution of actin filament
Membrane disruption:
Scratching cell monolayer by using sterile 18-gauge
needle.
Method of observation:
Confocal microscopy of disruption cell membrane at 15
seconds, 1 minute or 30 minutes after scratching.
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Disruption induces actin depolymerization
15s
30
1
FDx TRITC
Confocal imaging of wounded cells localized cortical domains of RGM1 cells
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Influence of Ca 2+on regulationof actin filament
Cellnumber
Cellnumber
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Examination of actin depolymerization agents
in resealing
Agents:DNase1 (DN-2: 2 g/ml; DN-20: 20g/ml),Phalloidin (Pha-2: 2g/ml; Pha-20: 20g/ml) ,
Japsplakinolide (Jasp: 3g/ml),
and cytochalasin B (CyB: 3g/ml).
Method:RGM1 cells were pretreated with depolymerization agents
before disruption.
Examination:Chemiluminesence assays of releasing of membrane-
impermeant cytosolic molecule (ATP) when cells were loaded
with agents above;
Microscopic images (cytochalasin B).
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Without cytochalasin B Cytochalasin B 3g/ml
Loss of cortical actin and stress fiberFailure resealingNearly complete resealing
The role of actin depolymerization agents in resealing
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Conclusions
- Resealing can be facilitated by manipulation of
filamentous actin dissolution
- Filamentous actin content is reduced depending
on Ca2+ level shortly (~15 seconds) after wounding
- Resealing can be inhibited by manipulation of
filamentous actin stabilization.
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