The importance of glycosphingolipid synthesis in MDCK 3D epithelial morphogenesis and proper primary cilium development

LABROUSSE, MARIANO ELISEO; MOSCA, JAZMÍN MARÍA; KRIVOCAPICH, CLARA; PESCIO, LUCILA GISELE

Rosario

Congreso; LIX Annual Meeting SAIB 2023; 2023

Madin-Darby canine kidney (MDCK) cells are a well-established cell line model for studying epithelial morphogenesis. MDCK cells can be cultured in 2D and 3D conditions, rendering, in both cases, apico-basolateral polarity. However, evidence shows that 3D culture is a better model to study morphogenesis, forming cysts with a central lumen surrounded by a single layer of polarized cells. Instead, 2D cultures form monolayers of polarized cells, without lumenogenesis. Previous results from our laboratory showed that glycosphingolipids have a central role in MDCK cell differentiation in 2D cultures. In this study, we describe a method to obtain MDCK cell cysts on a collagen matrix and analyze the effect of glycosphingolipid depletion in epithelial morphogenesis. We monitor the 3D cystogenesis by bright-field microscopy. The time course showed three stages: lumen establishing, lumen enlarging, and lumen maintenance. We hypothesize that inhibition of glycosphingolipid synthesis affects epithelial morphogenesis and primary cilium development. Since lumen generation occurs on day 4, we treated 3D cultures with D-PDMP, an inhibitor of glucosylceramide synthase, on day 3 and performed immunofluorescence assays on day 9. Labeling of the actin cytoskeleton with phalloidin-FITC showed cysts with normal lumen: a monolayer of polarized cells surrounding an open central lumen, but multiluminal, atypical lumen, no lumen, and small cysts were also found. Most of the cysts presented a normal lumen both in control and treated cells, but D-PDMP - treated cells showed a higher percentage of small cysts. An important stage in the polarization process is the formation of primary cilia from the apical membrane projecting into the lumen. Immunofluorescence for acetylated tubulin allowed primary cilia detection and length analysis when observed by confocal scanning microscopy. Mostly of the cysts with normal lumen showed primary cilia extending towards the lumen, but D-PDMP - treated cells displayed longer cilia than control cells. Previous studies indicated that non – fully differentiated cells have longer primary cilium when compared to fully differentiated cells, proposing that cilia length reflects the cell status. Our findings suggest that glycosphingolipids are essential for MDCK 3D epithelial morphogenesis and proper primary cilium development.