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

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Remodeling the Actin Cytoskeleton to Enhance Neurite Outgrowth

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  1. Remodeling the Actin Cytoskeleton to Enhance Neurite Outgrowth Elizabeth Furnish, Casey Cunningham, Josef Kas, Christine Schmidt University of Texas at Austin

  2. Goals • Determine relationship between gelsolin and neurite (axon) outgrowth in PC12 cells • Enhance neurite extension by overexpressing gelsolin • Understand cellular mechanisms behind nerve regeneration

  3. Nerve Regeneration

  4. Anatomy ofNerve Growth Cone microtubules actin filaments neurofilaments - +

  5. 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

  6. Role of Gelsolin ligand receptor plasma membrane gelsolin actin Figure adapted from D. Bray, Cell Movements, Garland Publishing, New York, 1992, p.344

  7. Previous Experimentswith Gelsolin • Gelsolin mircoinjection? • Gelsolin overexpression in fibroblasts leads to enhanced motility

  8. Previous Experimentswith Gelsolin Knockout mice • Slower migration in fibroblasts • Delayed retraction of filopodia in neurites • Increased damage from stroke • Reduced phagocytosis by neutrophils

  9. 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

  10. Western Analysis S chemiluminescent P 2º Ab HRP 1º Ab membrane gelsolin

  11. Gelsolin Upregulated in the Presence of NGF reg 3d 1d 5d 7d +50 ng/ml NGF

  12. Gelsolin Upregulated in the Presence of NGF ng gsn/ mg lysate +NGF Days

  13. Gelsolin Upregulation Insensitive to Concentration of NGF reg 3d 1d 5d 7d +25 ng/ml NGF +50 ng/ml NGF

  14. Gelsolin Upregulation Insensitive to Concentration of NGF ng gsn/ mg lysate +NGF Days

  15. 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

  16. 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

  17. Immunofluorescence 2º Ab fluorescent Phalloidin fluorescent 1º Ab fixed cell gelsolin actin

  18. Actin Filament Distributionand Colocalization with Gelsolin Gelsolin/Actin Actin Gelsolin +gsn 10 mm

  19. Gelsolin Overexpressedin +gsn Clone reg 3d 1d 5d 7d +50 ng/ml NGF mock +gsn

  20. Gelsolin Overexpressedin +gsn Clone ng gsn/ mg lysate +NGF Days

  21. 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

  22. 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

  23. Neurite Outgrowthat 3d +NGF mock +gsn 100 mm

  24. Gelsolin EnhancesNeurite Length Average Neurite Length (mm) wt mock +gsn p<0.01

  25. Video Microscopy of Neurite Outgrowth mock +gsn 100 mm

  26. 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

  27. 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

  28. Current Experiments • Determine pools of exogenous (rat) and transfected (human) gelsolin • Quantify gelsolin mRNA levels • Measure neurite elongation rates

  29. Gelsolin is a Key Protein for Nerve Regeneration

  30. 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

  31. 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

  32. Neurite Length for Various Clones

  33. Gelsolin Colocalization Actin Gelsolin Gelsolin/Actin wt +gsn

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