Remodeling the actin cytoskeleton to enhance neurite outgrowth
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Remodeling the Actin Cytoskeleton to Enhance Neurite Outgrowth. Elizabeth Furnish, Casey Cunningham, Josef Kas, Christine Schmidt University of Texas at Austin. Goals. Determine relationship between gelsolin and neurite (axon) outgrowth in PC12 cells

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Remodeling the actin cytoskeleton to enhance neurite outgrowth

Remodeling the Actin Cytoskeleton to Enhance Neurite Outgrowth

Elizabeth Furnish, Casey Cunningham, Josef Kas, Christine Schmidt

University of Texas at Austin


Goals
Goals Outgrowth

  • Determine relationship between gelsolin and neurite (axon) outgrowth in PC12 cells

  • Enhance neurite extension by overexpressing gelsolin

  • Understand cellular mechanisms behind nerve regeneration



Anatomy of nerve growth cone
Anatomy of OutgrowthNerve Growth Cone

microtubules

actin filaments

neurofilaments

-

+


Actin accessory proteins

gelsolin Outgrowth

Actin Accessory Proteins

contracting

stiffening

gelating

monomer

monomer

binding

filaments

bundling

severing

capping

Figure adapted from D. Bray, Cell Movements, Garland Publishing, New York, 1992, p.87


Role of gelsolin
Role of Gelsolin Outgrowth

ligand

receptor

plasma

membrane

gelsolin

actin

Figure adapted from D. Bray, Cell Movements, Garland Publishing, New York, 1992, p.344


Previous experiments with gelsolin
Previous Experiments Outgrowthwith Gelsolin

  • Gelsolin mircoinjection?

  • Gelsolin overexpression in fibroblasts leads to enhanced motility


Previous experiments with gelsolin1
Previous Experiments Outgrowthwith Gelsolin

Knockout mice

  • Slower migration in fibroblasts

  • Delayed retraction of filopodia in neurites

  • Increased damage from stroke

  • Reduced phagocytosis by neutrophils


Objectives
Objectives Outgrowth

  • Quantify gelsolin expressionlevels upon differentiation with NGF

  • Transfect with gelsolin gene, verify actin distribution, and monitor gelsolin expression levels

  • Determine effect of gelsolin overexpression on neurite extension


Western analysis
Western Analysis Outgrowth

S

chemiluminescent

P

2º Ab

HRP

1º Ab

membrane

gelsolin


Gelsolin upregulated in the presence of ngf
Gelsolin Upregulated in the Presence of NGF Outgrowth

reg

3d

1d

5d

7d

+50 ng/ml NGF


Gelsolin upregulated in the presence of ngf1
Gelsolin Upregulated in the Presence of NGF Outgrowth

ng gsn/

mg lysate

+NGF

Days


Gelsolin upregulation insensitive to concentration of ngf
Gelsolin Upregulation Insensitive to OutgrowthConcentration of NGF

reg

3d

1d

5d

7d

+25 ng/ml NGF

+50 ng/ml NGF


Gelsolin upregulation insensitive to concentration of ngf1
Gelsolin Upregulation Insensitive to OutgrowthConcentration of NGF

ng gsn/

mg lysate

+NGF

Days


Objectives1
Objectives Outgrowth

  • Quantify gelsolin expression levels upon differentiation with NGF

  • Transfect with gelsolin gene, verify actin distribution, and monitor gelsolin expression levels

  • Determine effect of gelsolin overexpression on neurite extension


Transfection methods
Transfection Methods Outgrowth

PC12 cells were transfected with

mock and gelsolin plasmids

SV-neo

SV-neo

Mock

10 kb

Gelsolin

12.5 kb

b-actin

b-actin

AmpR

AmpR

Gelsolin gene


Immunofluorescence
Immunofluorescence Outgrowth

2º Ab

fluorescent

Phalloidin

fluorescent

1º Ab

fixed cell

gelsolin

actin


Actin filament distribution and colocalization with gelsolin
Actin Filament Distribution Outgrowthand Colocalization with Gelsolin

Gelsolin/Actin

Actin

Gelsolin

+gsn

10 mm


Gelsolin overexpressed in gsn clone
Gelsolin Overexpressed Outgrowthin +gsn Clone

reg

3d

1d

5d

7d

+50 ng/ml NGF

mock

+gsn


Gelsolin overexpressed in gsn clone1
Gelsolin Overexpressed Outgrowthin +gsn Clone

ng gsn/

mg lysate

+NGF

Days


Objectives2
Objectives Outgrowth

  • Quantify gelsolin expression levels upon differentiation with NGF

  • Transfect with gelsolin gene, verify actin distribution, and monitor gelsolin expression levels

  • Determine effect of gelsolin overexpression on neurite extension


Migration analysis
Migration Analysis Outgrowth

2 dishes

per clone

2

1

6-10 pictures

per dish

1

2

1

2

4

4

3

6

3

6

5

5

median of each picture

average of medians


Neurite outgrowth at 3d ngf
Neurite Outgrowth Outgrowthat 3d +NGF

mock

+gsn

100 mm


Gelsolin enhances neurite length
Gelsolin Enhances OutgrowthNeurite Length

Average

Neurite

Length

(mm)

wt

mock

+gsn

p<0.01


Video microscopy of neurite outgrowth
Video Microscopy of Neurite Outgrowth Outgrowth

mock

+gsn

100 mm


Results and conclusions
Results and Conclusions Outgrowth

  • Gelsolin is upregulated upon differentiation with NGF

    • Key protein

    • Optimal level

  • Gelsolin upregulation is insensitive to changing concentration of NGF

    • NGF threshold


  • Results and conclusions1
    Results and Conclusions Outgrowth

    • +gsn clone overexpresses gelsolin,but is less sensitive to NGF

      • Already at optimal level

  • Average neurite length increased for +gsn clone

    • Gelsolin overexpression enhances neurite outgrowth


  • Current experiments
    Current Experiments Outgrowth

    • Determine pools of exogenous (rat) and transfected (human) gelsolin

    • Quantify gelsolin mRNA levels

    • Measure neurite elongation rates



    Acknowledgements

    Undergraduate Research Assistants Outgrowth

    Suzy Bowers

    Gabi Ramos

    Video Microscopy

    M.D. Anderson Cancer Center

    Dr. Charles Patrick, Jr.

    Eric Brey

    Acknowledgements

    • Funding Sources

      • NSF

      • Whitaker Foundation


    Western methods
    Western Methods Outgrowth

    • Whole cell lysate diluted and run on gel with gelsolin standards

    • Expose membrane to x-ray film, develop, and digitally scan for image analysis

    • Calibration curve previously determined to give linear correlation between OD of gelsolin bands and amount

    • Only analyze lanes with gelsolin levels within calibration curve

    Gsn 30ng 20ng 15ng

    20mg 15mg 10mg Gsn Clone


    Neurite length for various clones
    Neurite Length for OutgrowthVarious Clones


    Gelsolin colocalization
    Gelsolin Colocalization Outgrowth

    Actin

    Gelsolin

    Gelsolin/Actin

    wt

    +gsn


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