Akap350 Recruits Eb1 to The Spindle Poles, Ensuring Proper Spindle Orientation and Lumen Formation in 3d Epithelial Cell Cultures

Abstract

The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

Insane in the apical membrane: Trafficking events mediating apicobasal epithelial polarity during tube morphogenesis

“…Thus, it is conceivable that if the cells divide in the wrong plane, either due to misalignment of the microtubule spindle or cleavage furrow ingression direction, erroneous lumens may arise from each cell division. There are a number of proteins known to regulate spindle orientation during lumen expansion, including Cdc42 and its GEFs and GAPs 54–57 , polarity complex proteins 55,58 , phosphatidylinositol phosphatases 59 , microtubule binding proteins 60 , and other small GTPases 61 . Consequently, while apical membrane biogenesis is crucial for lumen formation, proper orientation of later cytokinesis events is also essential for expansion and maintenance of a single lumen.…”

The Small-Molecule Inhibitor MRIA9 Reveals Novel Insights into the Cell Cycle Roles of SIK2 in Ovarian Cancer Cells

“…Several lines of evidence indicated that the microtubule plus-end binding protein EB1 plays a critical role in defining the spindle position. Through mediating the interaction between microtubule ends, the cell cortex, and dynein motors, EB1 is involved in spindle assembly, dynamic, and positioning of the spindle [22][23][24]. Intriguingly, in our immunoprecipitation (IP) experiment using SIK2 antibodies, EB1 was found to precipitate with SIK2 in G2-synchronized SKOV-3 cells (Figure 3H).…”

A centrosomal view of CNS growth

“…Accordingly, the removal of mature centriole-specific proteins, including ninein (Wang et al, 2009), WDR62 and ASPM (Gai et al, 2016;Jayaraman et al, 2016), is sufficient to cause premature depletion of progenitor cells from the VZ and to impair CNS growth. Other proteins are recruited to the centrosome in a microtubule-independent manner by interacting with scaffold proteins, such as AKAP9 and pericentrin (Gillingham and Munro, 2000), which contain a localization domain (PACT domain) that targets the centrosome and serves to recruit structural and regulatory components such as γ-tubulin, microtubule binding proteins and signalling enzymes involved in microtubule nucleation (Almada et al, 2017). Supporting the relevance of centrosomal scaffold proteins in the control of the mode of NPC division, the removal of pericentrin triggers neurogenic divisions both in the chick spinal cord (Saade et al, 2017) and in the developing mouse cortex (Buchman et al, 2010).…”