Dr. Pedro J.I. Salas

Epithelial cell polarity and beyond

The People                             

Pedro J.I. Salas. M.D., Ph.D. - P.I.

Yolanda Figueroa - Lab. manager

      Flavia Wald, Ph.D. - Research Associate. Apical signaling in CACO-2 cells and the ezrin scaffold

Andrea Oriolo - Graduate Student. Apical localization of Microtubule Organizing Centers

     Amber Langshaw, M.D. - Pediatric Fellow. Molecular mechanisms of interaction between ezrin and keratins

Gisella Canessa - Rotation Graduate Student. Signaling regulation of Microtubule Organizing Center positioning.

   Anastasia Mashukova, Ph.D. - Research Associate. Signaling partners of apical keratins in epithelial cells.

The questions

CACO-2 cells viewed by confocal microscopy in the z axis (apical side up) showing colocalization of centrosomes (green, arrows) with intermediate filaments in interphase, but not in mitosis. Blue: DNA.

Our group is interested in how simple polarized epithelial cells acquire their polarity. Simple epithelia comprise, among others, the epithelia of the gut, liver, pancreas (and other exocrine glands), kidney, thyroid, etc. All these tissues form a single continuous layer of cells attached to the basement membrane and tightly bound to each other via intercellular junctions. At the top of those junctions there is the tight junction, immediately below the lumen of the organ, that forms a “belt” like structure around the top of the cell and separates the plasma membrane in two totally different domains: apical (in contact with the lumen, generally the external milieu) and basolateral, in contact with neighboring cells and the basement membrane (the internal milieu of the body). More specifically, therefore, the central question is how the apical domain gets organized in the first place. 

Polarization can be analyzed from three different perspectives: 

• Polarized membrane traffic, including sorting of membrane proteins at the trans-Golgi network and various endosomal compartments.
• Organization of the polarized cytoskeleton and submembrane scaffolds based on the cytoskeleton
• Polarized signaling, including signaling pathways that determine the apical versus basolateral domains, and polarized signaling molecules that create local phosphorylation gradients.

Techniques and protocols in the lab

Immunofluorescence and confocal microscopy (confocal facility)

Immunoelectron-microscopy, scanning, and transmission electron microscopy

Tissue culture, vectorial cell surface biotinylation

Immunoblot and immunoprecipitation

Protein-protein interactions

Molecular cloning and protein expression in bacteria, yeast, insect and mammalian cells

Funding

Our research is made possible by funding from the National Institute of Diabetes, Digestive and Kidney Diseases (RO1DK057805 , RO1DK076652), and by grants from the Department of Defense, the American Heart Association and the Crohn and Colitis Foundation of America.

Selected recent publications

- Ameen N.A., Figueroa Y., and Salas, P.J.I. 2001. Anomalous apical plasma membrane phenotype in CK8-deficient mouse enterocytes indicates a novel role of intermediate filaments in the polarization of simple epithelia. J. Cell Science 114(3):563-575.

- Figueroa Y., Wald F.A. and Salas P.J.I. 2002. p34cdc2 mediated phosphorylation mobilizes microtubule organizing centers from the apical intermediate filament scaffold in Caco-2 epithelial cells. J. Biol. Chem. 277(40):37848-54.

- Ameen N., Marino C., and Salas P.J.I.. 2003. cAMP stimulates physiologic exocytosis of CFTR to the apical surface in rat small intestinal epithelium. Am. J. Physiol. Cell Physiol..284:C429-C438.

- Wald F.A., Figueroa Y., Oriolo A.S., and Salas P.J.I.. 2003. Membrane repolarization is delayed in proximal tubules after ischemia/reperfusion: possible role of Microtubule-Organizing Centers. Am. J. Physiol. - Renal Physiol. . 285:F230-F240

- Wald, F.A., Oriolo A.S., Casanovas M.L., and Salas P.J.I. 2005. Intermediate filaments organize the apical ezrin scaffold in intestinal epithelial cells. Mol. Biol. of the Cell 16(9):4096-4107.

- Ramsauer V.P., Pino V., Carothers Carraway C.A., Salas P.J.I., and Carraway K.L.. 2006. Muc4-Erb2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand and chaperone for Erb2. Mol. Biol. of the Cell 17(7):2931-2941.

- Pino V, Ramsauer VP, Salas P.J., Carothers Carraway CA, and Carraway KL. 2006. Membrane Mucin Muc4 Induces Density Dependent Changes in Erk Activation in Mammary Epithelial and Tumor Cells: Role in Reversal of Contact Inhibition. J. Biol. Chemistry 281:29411-29420.

- Oriolo A.S., Wald F.A., Canessa G., and Salas P.J.I.. 2007. GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells. Mol. Biol. of the Cell  18:781-794.

- Oriolo A.S., Wald F.A., Ramsauer V.P., and Salas P.J.I.  2007. Intermediate filaments: a role in epithelial polarity. Exp. Cell Res., in press.