The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation

Abstract

SummaryMicrotubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.

New molecular insights into modulation of platelet reactivity in aspirin-treated patients using a network-based approach

“…CDC42 also plays a role in MAP kinase cascade, reinforcing a potential function of this pathway in PR modulation (Elvers 2015). Cytoskeleton-related proteins are also pointed out by this study, such as CDC42, coronin, spectrin and tropomyosin 3 which are known as actin-binding proteins (Barkalow et al 2003;Ge et al 2014;Pleines et al 2013), whereas MAPRE2 interacts with tubulin (Goldspink et al 2013). Upon activation, interactions between microtubules, actin and myosin cortex allows platelets to trigger shape change, starting with a disc to sphere transition and granule centering and ends up with secretion exocytosis (Sadoul 2015).…”

Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

“…Human TC7 colonic cells, which readily elongate and produce 10–12 µm tall cells with apico-basal arrays when grown to confluence, were used as a model to investigate the role of ninein in apico-basal MT array formation [22]. Ninein siRNA depletion was performed using previously tested sequences [8,15], which as expected [19] produced loss of centrosomal anchorage and disorganized MTs in non-confluent epithelial cells (figure 1 a , b ).…”

“…Small intestine was isolated and fractioned as previously described [22,71,72]. Isolated fractions were fixed in cold (−20°C) methanol or formaldehyde (9%)/methanol for 10 min and stained as above.…”

Microtubules Regulate Focal Adhesion Dynamics through MAP4K4

“…In contrast, EB2 does not play a direct role in MT dynamic instability (Komarova et al, 2009). Little is known about EB2’s cellular function (Goldspink et al, 2013). Our results raise the intriguing possibility that EB2 can act as an adaptor protein to recruit MAP4K4 to MTs, thus promoting FA turnover and cell motility.…”