Overexpression of Partner of Numb Induces Asymmetric Distribution of the PI4P 5-Kinase Skittles in Mitotic Sensory Organ Precursor Cells in Drosophila

Unequal segregation of cell fate determinants at mitosis is a conserved mechanism whereby cell fate diversity can be generated during development. In Drosophila, each sensory organ precursor cell (SOP) divides asymmetrically to produce an anterior pIIb and a posterior pIIa cell. The Par6-aPKC complex localizes at the posterior pole of dividing SOPs and directs the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized at the opposite pole. The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes. Here, we have examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs. Our analysis indicates that both Sktl and PIP2 reporters are uniformly distributed in mitotic SOPs. In the course of this study, we have observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP::PonLD) results in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. Our observation that Pon overexpression alters polar protein distribution is relevant because RFP::PonLD is often used as a polarity marker in dividing progenitors

PIP2 Reporters and GFP::Sktl localized uniformly in SOPs in the absence of RFP::Pon^LD.

<p>The distribution of PH::GFP (A), ENTH::GFP (B) and GFP::Sktl (C–D″) was examined by live imaging (A–C) or by antibody staining (anti-GFP in green and anti-Senseless in red in D–D″) in dividing SOPs at prometaphase. When RFP::Pon^LD was not co-expressed in SOPs, the PIP2 reporters and GFP::Sktl (panel C; 100%; n = 20) localized uniformly. All transgenes were expressed under the control of neur^P72Gal4Gal80^ts.</p

PIP2 reporters and GFP::Sktl localized asymmetrically in dividing SOPs expressing RFP::Pon^LD.

<p>The distribution of PH::GFP (A,A′), ENTH::GFP (B,B′) and GFP::Sktl (C,C′) was examined by live imaging (A–C″) or by antibody staining (anti-GFP in green in D–D″) in dividing SOPs expressing RFP::Pon^LD (A″,B″,C″,D″). Both PIP2 reporters and GFP::Sktl (C; 83%; n = 18) localize at the posterior pole of dividing SOPs at prometaphase when co-expressed with RFP::Pon^LD. All transgenes were expressed under the control of neur^P72Gal4 Gal80^ts. Anterior is on the right in this and all other figures.</p

GFP::Sktl localized at the posterior pole upon Pon overexpression.

<p>Distribution of PH::GFP (A), ENTH::GFP (B) and GFP::Sktl (C–F) in SOPs co-expressing Myc-tagged versions of full-length Pon (mycPon; A–D″), Numb (mycNumb; E) or Miranda (mycMira; F). Pon, Numb and Mira localized at the anterior pole of dividing SOPs at prometaphase (anti-Myc in red in D″; data not shown). Overexpression of Pon did not detectably modify the localization of PH::GFP (A), ENTH::GFP (B) in living SOPs. However, Pon overexpression resulted in the posterior accumulation of GFP::Sktl in live SOPs (panel C; 56%; n = 9) as well as in fixed cells (D,D′). Overexpression of Numb (E; 100%; n = 14) or Mira (F; 100%; n = 9) did not change the distribution of GFP::Sktl in live SOPs. All transgenes were expressed under the control of neur^P72Gal4Gal80^ts. (G) Quantification of the relative GFP signal intensity measured at the posterior <i>vs</i> anterior cortex of dividing SOPs at metaphase. GFP::Sktl was expressed in combination with the following constructs: 1. none (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003072#pone-0003072-g002" target="_blank">Fig. 2C</a>); 2. RFP::Pon^LD (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003072#pone-0003072-g001" target="_blank">Fig. 1C′</a>); 3. mycPon (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003072#pone-0003072-g003" target="_blank">Fig. 3C</a>); 4. mycNumb (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003072#pone-0003072-g003" target="_blank">Fig. 3E</a>); 5. mycMira (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003072#pone-0003072-g003" target="_blank">Fig. 3F</a>). Ratio values are (mean+/− standart deviation): 1.2 +/− 0.2; 2.0 +/− 0.4; 1.7 +/− 0.3; 1.4 +/− 0.2; 1.3 +/− 0.1. Expression of RFP::Pon^LD and mycPon, but not mycNumb nor mycMira, alters the distribution of GFP::Sktl in a statistically significant manner.</p

Endogenous Sktl localized uniformly in wild-type SOPs but accumulated at the posterior pole upon RFP::Pon^LD overexpression.

<p>Localization of endogenous Sktl (anti-Sktl in green) in wild-type (wt; A–A″) and RFP::Pon^LD-expressing SOPs (B–B″). RFP::Pon^LD was expressed under the control of neur^P72Gal4Gal80^ts. Sktl localized uniformly in wt mitotic SOPs (A,A′; 100%; n = 15), which were identified by Sens staining (red in A,A″). When RFP::Pon^LD was overexpressed in SOPs, Sktl localized asymmetrically at the posterior pole (B,B′; 69%; n = 32).</p