Search tips
Search criteria

Results 1-5 (5)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
Document Types
author:("octan, Asli")
1.  Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells 
Molecular Biology of the Cell  2012;23(12):2302-2318.
Ser-227 phosphorylation of Rab11-FIP2 by Par1b/MARK2 regulates the establishment of polarized epithelial monolayers in three-dimensional MDCK cell cultures and has an ongoing influence on the composition of both adherens and tight junctions in polarized epithelial cells.
The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.
PMCID: PMC3374749  PMID: 22553350
2.  Presynaptic α2δ-3 is required for synaptic morphogenesis independent of its Ca2+ - channel functions 
Nature neuroscience  2009;12(11):1415-1423.
Synaptogenesis involves the transformation of a growth cone into synaptic boutons specialized for transmitter release. In Drosophila embryos lacking the α2δ-3 subunit of presynaptic, voltage-dependent Ca2+ channels, we find that motor neuron terminals fail to develop synaptic boutons and cytoskeletal abnormalities arise, including the loss of ankyrin2. Nevertheless, functional presynaptic specializations are present and apposed to clusters of postsynaptic glutamate receptors. Heretofore, the α2δ-3 protein has been thought to function strictly as an auxiliary subunit of the Ca2+ channel, but the phenotype of α2δ-3 mutations cannot be explained by a channel defect: embryos lacking the pore-forming α1 subunit cacophony form boutons. The synaptogenic function of α2δ-3 requires only the α2 peptide, whose expression suffices to rescue bouton formation. Our results indicate that α2δ proteins have functions independent of their roles in the biophysics and localization of Ca2+ channels, and synaptic architecture depends on these novel functions.
PMCID: PMC2996863  PMID: 19820706
3.  Transcytosis of Polymeric Immunoglobulin A in Polarized Madin-Darby Canine Kidney Cells 
The transcytotic pathway allows for the bidirectional transport of endocytosed solutes, lipids, and proteins between the two membrane domains of polarized epithelial cells while maintaining the functional integrity of the epithelial tissue. A method is described to measure basolateral-to-apical transcytosis of immunoglobulin A (IgA) in polarized Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor (pIgR). The cells are grown on porous Transwell filter supports, and radiolabeled 125I-immunoglobulin A (IgA) is internalized from the basolateral pole of MDCK cells. During a subsequent 2-h chase, the amount of 125I-IgA that is recycled, degraded, or transcytosed is quantified. This assay can be adapted to follow the postendocytic fate of other 125I-labeled ligands and proteins.
PMCID: PMC2603048  PMID: 18369944
Apical; basolateral; immunoglobulin A (IgA); iodination; polarized Madin-Darby canine kidney (MDCK) cells; polymeric immunoglobulin receptor (pIgR); transcytosis
4.  Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells 
Molecular Biology of the Cell  2007;18(10):3978-3992.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
PMCID: PMC1995710  PMID: 17686995
5.  MARK2/EMK1/Par-1Bα Phosphorylation of Rab11-Family Interacting Protein 2 Is Necessary for the Timely Establishment of Polarity in Madin-Darby Canine Kidney Cells 
Molecular Biology of the Cell  2006;17(8):3625-3637.
Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Bα (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.
PMCID: PMC1525241  PMID: 16775013

Results 1-5 (5)