Mechanisms Behind Establishing Polarity of Asymmetric Cell Division in Drosophila Melanogaster Neuroblasts Amanda C. Baker Lab: Ken Prehoda Mentor: Mike.

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Presentation transcript:

Mechanisms Behind Establishing Polarity of Asymmetric Cell Division in Drosophila Melanogaster Neuroblasts Amanda C. Baker Lab: Ken Prehoda Mentor: Mike Drummond Department of Chemistry/Institute of Molecular Biology SPUR Symposium 8/20/10

Cellular Differentiation Is a Crucial Component of Development

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 RNAi 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 Chiharu Doug Michelle Marisa Jon Chris Ryan Nick Sasha Colin Evan Meagan SPUR Peter O’Day Blakely Strand Carson Dining Hall