an actin barrier for resealing

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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an actin barrier for resealing

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