659A Poster - 10. Cell biology: Cytoskeleton, organelles and trafficking
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Kristen Browder ^1,2; Seung-kyu Lee ^1,3; Elizabeth Klipfell ¹; Mark Travor ¹; Katelyn Wolfgang ¹; Claire Thomas ¹

Affiliations:
1) Penn State, University Park, PA; 2) Genentech, South San Francisco, CA; 3) National Institute of Aging (NIH/NIA/IRP), Baltimore, MD

Keywords:
b. cell polarity; a. cytoskeleton

Spectrin is a large F-actin crosslinking protein that most famously forms 2D networks in association with the plasma membrane of red blood cells. This ‘membrane skeleton’ confers cell shape and membrane strength during the rigors of circulation. In NON-erythroid tissues, spectrin has additional roles in the endomembrane system. We have previously shown that the apically polarized β_H‑spectrin (β_H), encoded by the karst locus in Drosophila, is required for the stability of several apical proteins, through the promotion of endosomal recycling to the plasma membrane - so called, ‘dynamic protein stabilization’. The apical protein determinant Crumbs recruits β_H to the apical membrane and is itself trafficked in a β_H-dependent manner. β_H binds to the Hippo/Warts pathway (HWP) regulator Expanded, which mediates Crumbs crosstalk to the HWP.
Here we report that a yeast 2-hybrid (Y2H) screen identified the PP2A substrate-specificity subunit Waldorf (a PP2A-PR72/B’’ isoform) to be a binding partner of β_H‑spectrin. Waldorf binds to β_H via a short, conserved sequence in its C-terminal globular domain. Genetic interaction and molecular epistasis experiments strongly suggest that PP2A with the Waldorf specificity-subunit bound to it (PP2A^Waldorf) acts as a negative regulator of Crumbs by acting to displace aPKC from Crumbs. Consistent with this notion, mutant versions of Crumbs lacking target residues for aPKC in the FERM-domain binding site do not respond to changes in Waldorf levels. Knockdown of Waldorf leads to reduced growth suggesting that Crumbs crosstalk with the HWP is a primary target of this homeostatic regulation. Overexpression of Waldorf specifically destroys the apical domain suggesting that Waldorf may also play a role in modulating the apical domain.
We also show that Waldorf modulates protein trafficking in a similar way to β_H and its previously reported partner Annexin B9 in that knockdown of Waldorf leads to an increase in Rab7-positive and acidic compartments, suggesting that PP2A^Waldorf also normally acts by suppressing lysosomal trafficking, most likely in favor of recycling pathways.
Our results support a model in which Crumbs recruits β_H in a complex with the HWP activator Expanded, and PP2A^Waldorf bound to β_H acts in a homeostatic fashion to limit Crumbs activation of the HWP by displacing aPKC. This in turn limits the amount of growth suppression caused by Crumbs-dependent HWP activation.