(M) Nedd1 localization in the crypt and villus (top). microtubule disorganization upon loss of CAMSAP3/Nezha. These data demonstrate that enterocyte microtubules have important roles in organelle organization but are not essential for growth under homeostatic conditions. INTRODUCTION The past two decades have provided significant insight into microtubule-binding proteins and their effects on microtubule dynamics and organization RFC37 in cultured cells. The roles of microtubules, as well as their organization and dynamics, in intact tissues are less well-understood (Muroyama and Lechler, 2017a ). In most differentiated cells, including the intestine, microtubules adopt noncentrosomal organizations, but we know little about how these networks form or their in vivo functions. The intestinal epithelium is a highly proliferative and polarized tissue. Proliferation is restricted to crypts, which are invaginations of the epithelium into the underlying mesenchyme (Tan and Barker, Nanaomycin A 2014 ). Crypt cells give rise to differentiated enterocytes, goblet cells, and enteroendocrine cells that populate the villus. Enterocytes, the most abundant of these, are columnar epithelia with an Nanaomycin A essential role in nutrient digestion, absorption, and transport. Prior work on microtubule function within the intestinal epithelium relied on cultured cells, such as Caco-2, or drug treatment of intestinal explants (Hugon plane. Scale = 5 m. (H) Mapping centriole position within the plane in villar cells. = 146 centrosomes. (I) Stitched image of a single crypt-villus axis showing CDK5RAP2 localization. Scale = 25 m. (J) Quantification of CDK5RAP2 fluorescence intensity along the crypt-villus axis. (K) CDK5RAP2 and pericentrin localization in the crypt and villar cells. Scale = 10 m. (L) Stitched image of a single crypt-villus axis showing Nedd1 localization. Scale = 25 m. (M) Nedd1 localization in the crypt and villus (top). Scale = 10 m. Bottom panels show the zoomed region on the apical surface of the crypt, where Nedd1 is colocalized with pericentrin and also forms noncentrosomal clusters at the apical surface. White arrows indicate pericentrin foci. Scale = 5 m. (N) -Tubulin localization in the intestinal crypt and villus. Scale = 10 m. All dotted lines indicate basement membrane. In villi, microtubules formed apicalCbasal arrays that were highly enriched on the apical side of the nucleus, with a few microtubules extending to the basal surface (Figure 1, C and D). This is consistent with previous reports in simple columnar epithelial cells (Troutt and Burnside, 1988 ; Bacallao plane of the cell when viewed from above (Figure 1, G and H). Although centrioles were intact in all cells, we noted a striking reduction of pericentriolar material (PCM) between crypts and villi. CDK5RAP2, a pericentriolar protein that promotes -TuRC nucleation activity, was robustly associated with Nanaomycin A apical puncta in crypts. In contrast, villar cells had very low levels of CDK5RAP2 at centrosomes (Figure 1, ICK). Pericentrin showed a similar localization pattern, suggesting that the pericentriolar material is largely lost upon differentiation (Figure 1K). To test this, we examined two additional PCM proteins, -tubulin and Nedd1. In crypts, both Nedd1 and -tubulin were associated with apical puncta. These puncta colocalized with pericentrin, but both also had a diffuse apical localization in addition to their centrosomal localization. In villar cells, there was negligible Nedd1 and -tubulin associated with Nanaomycin A centrosomes. Instead, these proteins were found associated with the apical side of the cell. This relocalization of -tubulin has been noted before and is consistent with MTOC activity shifting from the centrosome to the apical cortex where microtubule minus ends are tethered (Salas, 1999 ). It is notable that Nedd1 demonstrates a similar reorganization as -TuRC because Nedd1/-TuRC complexes have been implicated in anchoring microtubule minus ends in keratinocytes (Muroyama = 30 cells from each of two mice for each genotype. Error bars show SD. (G) Quantification of HA-positive villar.
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