PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in theDrosophilaoocyte

Abstract

The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fatedetermining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.

Novel Genetic Approach for In Vivo Vascular Imaging in Mice

“…Costaining for the BirA-specific HA epitope tag and the endoplasmic reticulum (ER) marker Calnexin showed that BirA was correctly targeted to the ER (Figure 2A). The Myc tag integrated in the Biotag protein colocalized with Phaloidin staining for F-actin, which largely underlies the membrane 19 (Figure 2B). Finally, costaining for both the HA and Myc tags showed partially overlapping expression patterns, confirming that BirA and Biotag colocalize in the ER as the Biotag protein is being trafficked to the cell membrane (Figure 2C).…”

Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila

“…Instead, our data suggest that establishment of spermatid cyst polarity relies on localized recruitment of the exocyst, which drives targeted membrane delivery to the growing end. Alternatively, or in addition, PIP 2 and the exocyst may help establish the axis of polarization by linking the spermatid nuclear envelope to the plasma membrane, as suggested from analysis of sktl 2.3 mutant clones during oogenesis (Gervais et al , 2008) or by regulating formation of the flagellar membrane that surrounds the growing end of the axoneme (Fritz-Niggli and Suda, 1972; Tokuyasu, 1975). Indeed, given the structural similarity between flagella and cilia and the requirement for the exocyst in ciliogenesis, PIP 2 could play a conserved role in regulating this critical process.…”

“…Unc-GFP (Baker et al , 2004) flies were generously provided by James Baker and Maurice Kernan (SUNY Stony Brook, Stony Brook, NY). s ktl 2.3 is a hypomorphic allele of sktl that affects polarity during Drosophila oogenesis (Gervais et al , 2008). Clones of germ cells mutant for s ktl 2.3 were generated in flies of genotype w , hsFLP/Y; FRT42B, sktl 2.3 /FRT42B, and Ubi-GFP.nls using the FLP-FRT (fragment length polymorphism and recognition target system, respectively; Golic and Lindquist, 1989).…”

Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte

“…Decoration of the nuclear envelope was achieved with a tagged β–importin (Fs(2)Ket-GFP), which interacts and co-localizes with nucleoporins22, while the plasma membrane was labelled by the PIP2-binding domain of the phospholipase- δ1 tagged with RFP (PH PLC-∂1 -RFP)2324 (Fig. 1a,b).…”

“…Interestingly, several components of the plasma membrane and its associated cortex are required for proper nuclear positioning in the oocyte. The PAR proteins PAR-3 and PAR-1, as well as the phosphoinositide PIP2, are necessary for nuclear anchorage to the plasma membrane2342.…”