Par3 Controls Epithelial Spindle Orientation by aPKC-Mediated Phosphorylation of Apical Pins

Abstract

Summary Background Formation of epithelial sheets requires that cell division occurs in the plane of the sheet. During mitosis, spindle poles align so the astral microtubules contact the lateral cortex. Confinement of the mammalian Pins protein to the lateral cortex is essential for this process. Defects in signaling through Cdc42 and atypical protein kinase C (aPKC) also cause spindle misorientation. When epithelial cysts are grown in 3D cultures, mis-orientation creates multiple lumens. Results We now show that silencing of the polarity protein Par3 causes spindle mis-orientation in MDCK cell cysts. Silencing of Par3 also disrupts aPKC association with the apical cortex, but expression of an apically-tethered aPKC rescues normal lumen formation. During mitosis, Pins is mislocalized to the apical surface in the absence of Par3, or by inhibition of aPKC. Active aPKC increases Pins phosphorylation on Ser401, which recruits 14-3-3 protein. 14-3-3 binding inhibits association of Pins with Gαi, through which Pins attaches to the cortex. A Pins S401A mutant mislocalizes over the cell cortex and causes spindle orientation and lumen defects. Conclusions The Par3/aPKC polarity proteins ensure correct spindle pole orientation during epithelial cell division by excluding Pins from the apical cortex. Apical aPKC phosphorylates Pins, which results in the recruitment of 14-3-3 and inhibition of binding to Gαi, so the Pins falls off the cortex. In the absence of a functional exclusion mechanism, astral microtubules can associate with Pins over the entire epithelial cortex, resulting in randomized spindle pole orientation.

Symmetrical and asymmetrical division analysis provides evidence for a hierarchy of prostate epithelial cell lineages

“…Previous studies have indicated that polarization in stem cells is necessary for asymmetrical divisions 14,15 . However, other studies reported that polarization that is associated with tight junctions or adherens junctions promotes symmetrical divisions [27][28][29] . To determine the polarization patterns of the prostate epithelium, we performed double immunofluorescence of prostate sections with p63, CK8 and antibodies against the polarization molecules Par3 and aPKC.…”

“…Cell polarity is one important feature of the epithelium 49 and has been shown to affect cell division modes 14,27 and tumorigenesis 50,51 . Our study indicates that the different expression patterns of the Par3 protein are observed in luminal and basal cells (Supplementary Fig.…”

FAK transduces extracellular forces that orient the mitotic spindle and control tissue morphogenesis

“…5d,e). Staining with the tight junction marker ZO-1 revealed that apicobasal polarity is not affected in FAK morphants, whereas other polarized molecules involved in spindle orientation like the Par6/3-aPKC complex also remain properly localized even in cells with completely misoriented spindles 45,46 ( Supplementary Fig. 6a-c).…”

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

“…For example, erratic alignment of the mitotic spindle could enable efficient expansion of a cell mass in every direction, thus promoting hyperplasia. Out-of-alignment mitotic spindle may also increase aneuploidy [5,23]. Altered cell adhesion and extracellular matrix-dependent signalling mechanisms may make cells more migratory [24], loss of lipid asymmetry may deregulate spatial PI3K signalling and any cell-intrinsic (e.g.…”

“…The molecular asymmetries guide directional exocytosis and secretion of digestive enzymes from pancreas, milk from mammary epithelium and vectorial transfer of nutrients across the gut epithelium to blood [1]. Furthermore, extension of epithelial monolayers requires that cell divisions occur in the plane of the sheet and many key cell polarity proteins are involved in determining alignment of the mitotic spindle so that it is perpendicular to the axis of apico-basal polarity [4][5][6]. In addition to many proteins, lipids are also asymmetrically distributed in polarized cells [1].…”