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
  • 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 ofNerve Growth Cone

microtubules

actin filaments

neurofilaments

-

+

actin accessory proteins

gelsolin

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

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 Experimentswith Gelsolin
  • Gelsolin mircoinjection?
  • Gelsolin overexpression in fibroblasts leads to enhanced motility
previous experiments with gelsolin1
Previous Experimentswith Gelsolin

Knockout mice

  • Slower migration in fibroblasts
  • Delayed retraction of filopodia in neurites
  • Increased damage from stroke
  • Reduced phagocytosis by neutrophils
objectives
Objectives
  • 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

S

chemiluminescent

P

2º Ab

HRP

1º Ab

membrane

gelsolin

objectives1
Objectives
  • 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

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

2º Ab

fluorescent

Phalloidin

fluorescent

1º Ab

fixed cell

gelsolin

actin

gelsolin overexpressed in gsn clone
Gelsolin Overexpressedin +gsn Clone

reg

3d

1d

5d

7d

+50 ng/ml NGF

mock

+gsn

gelsolin overexpressed in gsn clone1
Gelsolin Overexpressedin +gsn Clone

ng gsn/

mg lysate

+NGF

Days

objectives2
Objectives
  • 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

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

gelsolin enhances neurite length
Gelsolin EnhancesNeurite Length

Average

Neurite

Length

(mm)

wt

mock

+gsn

p<0.01

results and conclusions
Results and Conclusions
  • 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
  • +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
  • Determine pools of exogenous (rat) and transfected (human) gelsolin
  • Quantify gelsolin mRNA levels
  • Measure neurite elongation rates
acknowledgements
Undergraduate Research Assistants

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
  • 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

gelsolin colocalization
Gelsolin Colocalization

Actin

Gelsolin

Gelsolin/Actin

wt

+gsn

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