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??

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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