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
Cellular Differentiation Is a Crucial Component of Development
www.bioscience.org
Protein Polarity is Vital for the Proper Differentiation of Cells
Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate
Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate•Phosphorylated Miranda becomes cytoplasmic
Apical aPKC Localization Is Key For Normal Polarity
Doe 2009
Miranda
•aPKC phosphorylates Miranda at the metaphase plate•Phosphorylated Miranda becomes cytoplasmic
How does aPKC get to the apical cortex??
boards.ign.com
Cdc42 is Required for Apical aPKC Restriction
Cdc42 is a key regulator of polarity during asymmetric cell division.
• Small Rho GTPase• Cycles between GTP and GDP bound states
Miranda
GTP-ase Activating Proteins (GAPs)
Silencing RhoGAP102a via RNAi Causes aPKC to Extend its Apical
Domain
Methods
• Genetic Crosses
Methods
• Genetic Crosses• Larval Incubation
Methods
• Genetic Crosses• Larval Incubation• Antibody Staining
Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy
Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy • Photoshop
Methods
• Genetic Crosses• Larval Incubation• Antibody Staining• Confocal Microscopy • Photoshop • Image J
Main Question
• Is RhoGAP102a required for apical aPKC restriction?
Experiment: Silence RhoGAP102a via RNAi
aPKC Is No Longer Restricted to the Apical Cortex
RhoGAP102a RNAiaPKC/PH3 Miranda DPN Merge
aPKC/PH3 Miranda DPN Merge
Wild Type
aPKC Basal/Apical Ratios
• P-Value=0.0130
Does Par6 Exhibit the Same Affect as aPKC?
Par6 Can Also Be Detected in the Basal Cortex
aPKC/PH3 Miranda Par6 Merge
aPKC/PH3 Par6 Miranda Merge
Par-6 Basal/Apical Ratios
• P-Value=0.008
Does Bazooka Exhibit the Same Affect as aPKC?
Bazooka Localization in RhoGAP102a RNAi is Wildtype
aPKC/PH3 Bazooka DPN Merge
Bazooka Basal/Apical Ratio
P-Value=0.6165
Conclusion
• RhoGAP102a is required for the proper apical restriction of aPKC.
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)
Special Thanks!Ken Mike ChiharuDougMichelleMarisa JonChrisRyanNickSashaColinEvanMeagan
SPURPeter O’DayBlakely Strand Carson Dining Hall