mechanisms behind establishing polarity of asymmetric cell division in drosophila melanogaster...
TRANSCRIPT
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Mechanisms Behind Establishing Polarity of Asymmetric Cell Division in
Drosophila Melanogaster NeuroblastsAmanda C. BakerLab: Ken Prehoda
Mentor: Mike DrummondDepartment of Chemistry/Institute of Molecular Biology
SPUR Symposium 8/20/10
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Cellular Differentiation Is a Crucial Component of Development
www.bioscience.org
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Protein Polarity is Vital for the Proper Differentiation of Cells
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Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
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Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate
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Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate•Phosphorylated Miranda becomes cytoplasmic
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Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate•Phosphorylated Miranda becomes cytoplasmic
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How does aPKC get to the apical cortex??
boards.ign.com
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Cdc42 is Required for Apical aPKC Restriction
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Cdc42 is a key regulator of polarity during asymmetric cell division.
• Small Rho GTPase• Cycles between GTP and GDP bound states
Miranda
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GTP-ase Activating Proteins (GAPs)
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Silencing RhoGAP102a via RNAi Causes aPKC to Extend its Apical
Domain
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Methods
• Genetic Crosses
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Methods
• Genetic Crosses• Larval Incubation
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Methods
• Genetic Crosses• Larval Incubation• Antibody Staining
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Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy
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Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy • Photoshop
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Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy • Photoshop • Image J
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Main Question
• Is RhoGAP102a required for apical aPKC restriction?
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Experiment: Silence RhoGAP102a via RNAi
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aPKC Is No Longer Restricted to the Apical Cortex
RhoGAP102a RNAiaPKC/PH3 Miranda DPN Merge
aPKC/PH3 Miranda DPN Merge
Wild Type
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aPKC Basal/Apical Ratios
• P-Value=0.0130
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Does Par6 Exhibit the Same Affect as aPKC?
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Par6 Can Also Be Detected in the Basal Cortex
aPKC/PH3 Miranda Par6 Merge
aPKC/PH3 Par6 Miranda Merge
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Par-6 Basal/Apical Ratios
• P-Value=0.008
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Does Bazooka Exhibit the Same Affect as aPKC?
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Bazooka Localization in RhoGAP102a RNAi is Wildtype
aPKC/PH3 Bazooka DPN Merge
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Bazooka Basal/Apical Ratio
P-Value=0.6165
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Conclusion
• RhoGAP102a is required for the proper apical restriction of aPKC.
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Further Directions
• Isolate RhoGAP102a via PCR• Determine if there is a direct Cdc42-
RhoGAP102a interaction using a coimmunoprecipitation assay
• In vitro GTPase Assay• Construct CRIB epitope tag in order to
visualize GTP bound Cdc42 (Mike)
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Special Thanks!Ken Mike ChiharuDougMichelleMarisa JonChrisRyanNickSashaColinEvanMeagan
SPURPeter O’DayBlakely Strand Carson Dining Hall