Exocyst Is Involved in Cystogenesis and Tubulogenesis and Acts by Modulating Synthesis and Delivery of Basolateral Plasma Membrane and Secretory Proteins

Abstract

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

Intracellular signaling via ERK/MAPK completes the pathway for tubulogenic fibronectin in MDCK cells

“…Branching morphogenesis of tubular epithelium is a common and important feature of vertebrate organogenesis [1,22,32]. The MAPK/ERK pathway, which is downstream of receptor tyrosine kinases, leads to the phosphorylation, and hence activation, of ERK and has been shown to be important in branching morphogenesis in several systems, including the Drosophila trachea [20] and murine salivary gland [21].…”

“…Recombinant HGF at 100 ng/ml was added to the basolateral compartment of MDCK cell monolayers following a 1 h pretreatment either with or without U0126 (Promega) or PD098059 (Sigma), both inhibitors of MEK, at 10 μM and 50μM respectively. HGF at 100 ng/ml has been shown to induce tubulogenesis in MDCK cells [1,19]. Recombinant human HGF was generously provided by the late R. Schwall (Genentech, South San Francisco).…”

“…Epithelial organs such as the kidney, lung, and salivary gland develop from a process termed branching morphogenesis [1]. Though branching morphogenesis is incompletely understood, genes involved in this process are conserved in different organs, and indeed throughout evolution.…”

“…The vast majority of studies examining cyst and tubule formation using MDCK cells were performed with Strain II cells [1,11,12]. MDCK Strain I cells, derived from an early passage of the cell population, and MDCK Strain II cells, which predominate in later passages, originated from separate nephron segments [13,14].…”

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

“…LDLR fusion was shown at this site by time-lapse confocal microscopy (Kreitzer et al, 2003), and Shrew-1 and VSV-G fusion was found at this site by tannic acid-blocking experiments (Polishchuk et al, 2004;Jakob et al, 2006). In polarized MDCK cells, the Sec6/8 complex is recruited to (Yeaman et al, 2004) and localized at the apical junctional complex along the lateral membrane (Grindstaff et al, 1998;Lipschutz et al, 2000), which is recognized to be an area of active exocytosis (Kreitzer et al, 2003). We previously reported that Naked2-associated vesicles accumulated asymmetrically at the basolateral corner of MDCK cells stably expressing myristoylation-deficient G2A Naked2.…”