- Greater New York City Area
Ryan Schreiner's Overview
Ryan Schreiner's Publications
Beta Cyclodextrins Bind, Stabilize and Remove Lipofuscin Bisretinoids from Retinal Pigment Epithelium.
Authors: Marcelo Nociari, Guillermo Lehmann Mántaras, Andres Perez Bay, MD/PhD, RA Rada, Z Jiang, S Goicochea, Ryan Schreiner, J David Warren, Jufang Shan, SA de Beaumais, M Menand
- March 2014
Manuscript Accepted, will be online shortly.
- Developmental Cell
- November 2013
In this issue of Developmental Cell, a Matters Arising by Juan Bonifacino’s laboratory (Guo et al., 2013) addresses an emerging topic in epithelial cell biology, the complementary roles of the clathrin adaptors AP-1A and AP-1B in basolateral trafficking in epithelia. AP-1A and AP-1B are twin tetrameric clathrin adaptors; AP-1A is ubiquitous, whereas AP-1B is expressed only by epithelial cells. They share three subunits (β1, σ1, and γ) but differ in the possession of different (albeit 80% homologous) medium subunits (μ1A and μ1B). The role of AP-1B in basolateral trafficking was established over a decade ago (Fölsch et al., 1999), whereas the participation of AP-1A in basolateral trafficking was only demonstrated last year, in two collaborative papers between Bonifacino’s laboratory and our laboratory, one of them in Developmental Cell (Carvajal-Gonzalez et al., 2012,Gravotta et al., 2012). Therefore, the details of how they complement each other in basolateral sorting are still unclear. Guo et al. now postulate a model in which the two adaptors are paralogs with identical localization and function that differ in the repertoire of proteins that they can sort.
The Kinesin KIF16B mediates Apical Transcytosis of Transferrin Receptor in AP-1B deficient epithelia.
Authors: Andres Perez Bay, MD/PhD, Ryan Schreiner, Francesca Mazzoni, Carvajal-Gonzalez JM, Gravotta D, Perret E, Guillermo Lehmann Mántaras, Zhu YS, Rodriguez-Boulan, E
- EMBO Journal
- July 31, 2013
Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.
Authors: Jin Xu, Kimberly A Toops, Fernando Diaz, Jose Maria Carvajal Gonzalez, Diego Gravotta, Francesca Mazzoni, Ryan Schreiner, Enrique Rodriguez-Boulan, Aparna Lakkaraju
- Journal of Cell Science
- October 4, 2012
Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigated the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain, and in cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia, and suggest that AP-1 is a critical regulator of this process.
Basolateral sorting of CAR through interaction of a canonical YXXΦ motif with the clathrin adaptors AP-1A and AP-1B.
Authors: Carvajal-Gonzalez JM, Gravotta D, Mattera R, Fernando Diaz, Andres Perez Bay, MD/PhD, Roman AC, Ryan Schreiner, Thuenauer R, Bonifacino JS, Rodriguez-Boulan E
- Proceedings of the National Academy of Sciences
- March 6, 2012
The coxsackie and adenovirus receptor (CAR) plays key roles in epithelial barrier function at the tight junction, a localization guided in part by a tyrosine-based basolateral sorting signal, (318)YNQV(321). Sorting motifs of this type are known to route surface receptors into clathrin-mediated endocytosis through interaction with the medium subunit (μ2) of the clathrin adaptor AP-2, but how they guide new and recycling membrane proteins basolaterally is unknown. Here, we show that YNQV functions as a canonical YxxΦ motif, with both Y318 and V321 required for the correct basolateral localization and biosynthetic sorting of CAR, and for interaction with a highly conserved pocket in the medium subunits (μ1A and μ1B) of the clathrin adaptors AP-1A and AP-1B. Knock-down experiments demonstrate that AP-1A plays a role in the biosynthetic sorting of CAR, complementary to the role of AP-1B in basolateral recycling of this receptor. Our study illustrates how two clathrin adaptors direct basolateral trafficking of a plasma membrane protein through interaction with a canonical YxxΦ motif.
Basolateral Sorting Signals Regulating Tissue-Specific Polarity of Heteromeric Monocarboxylate Transporters in Epithelia.
Authors: Castorino J, Deborde S, Ami Deora, Ryan Schreiner, Gallagher-Colombo S, Rodriguez-Boulan E, Nancy Philp
- April 2011
Many solute transporters are heterodimers composed of non-glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue-specific functions. To date, the mechanisms regulating the tissue-specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton-coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C-terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin-dependent but clathrin adaptor AP1B-independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue-specific polarity.
Authors: Ryan Schreiner, Frindt G, Fernando Diaz, Carvajal-Gonzalez JM, Andres Perez Bay, MD/PhD, Palmer LG, Vladimir Marshansky, Brown D, Nancy Philp, Enrique Rodriguez-Boulan
- Kidney International
- August 2010
It is well established that many cognate basolateral plasma membrane proteins are expressed apically in proximal tubule cells thus optimizing the reabsorption capacity of the kidney. The protein clathrin and its adapter proteins normally regulate basolateral polarity. Here we tested whether the unique proximal tubule polarity is dependent on an epithelial-specific basolateral clathrin adapter, AP1B, present in most other epithelia. Quantitative PCR of isolated mouse renal tubules showed that AP1B was absent in proximal tubules but present in medullary and cortical thick ascending limbs of Henle, and cortical collecting ducts. Western blot confirmed the absence of AP1B in three established proximal tubule cell lines. Knockdown of AP1B by shRNA in prototypical distal tubule MDCK cells resulted in redistribution of the basolateral parathyroid hormone receptor, the insulin-like growth factor II receptor/calcium-independent mannose-6-phosphate receptor, and the junctional adhesion molecule, JAM-C, to a proximal tubule-like nonpolar localization. Yeast two-hybrid assays detected direct interactions between the cytoplasmic tails of these plasma membrane proteins and the cargo-binding region of the AP1B complex. Hence, our results show that differential expression of AP1B contributes to normal kidney function and illustrates possible roles of this adapter protein in kidney development, physiology, and pathology.
Authors: Gravotta D, Ryan Schreiner, Schoggins J, Falck-Pedersen E
- Proceedings of the National Academy of Sciences
- July 7, 2009
Adenoviruses invading the organism via normal digestive or respiratory routes require the Coxsackie-adenovirus receptor (CAR) to infect the epithelial barrier cells. Because CAR is a component of tight junctions and the basolateral membrane and is normally excluded from the apical membrane, most epithelia are resistant to adenoviruses. However, we discovered that a specialized epithelium, the retinal pigment epithelium (RPE), anomalously expressed CAR at the apical surface and was highly susceptible to adenovirus infection. These properties of RPE cells correlated with the absence of the epithelial-specific clathrin adaptor AP1B. Furthermore, knockdown of this basolateral sorting adaptor in adenovirus-resistant MDCK cells promoted apical localization of CAR and increased dramatically Adenovirus infectivity. Targeting assays showed that AP1B is required for accurate basolateral recycling of CAR after internalization. AP1B knock down MDCK cells missorted CAR from recycling endosomes to the apical surface. In summary, we have characterized the cellular machinery responsible for normal sorting of an adenovirus receptor and illustrated how tissue-specific variations in such machinery result in drastic changes in tissue-susceptibility to adenoviruses.
LIM kinase 1 and cofilin regulate actin filament population required for dynamin-dependent apical carrier fission from the trans-Golgi network.
Authors: Salvarezza S, Sylvie Deborde, Ryan Schreiner, Campagne, F, Kessels, M. M, Qualmann, B, Caceres, A, Kreitzer, G, Enrique Rodriguez-Boulan
- Molecular Biology of the Cell
- January 2009
The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin-dependent generation and/or fission of precursors to p75 transporters.
Authors: Sylvie Deborde, Perret E, Gravotta D, Ami Deora, Salvarezza S, Ryan Schreiner, Enrique Rodriguez-Boulan
- April 10, 2008
Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.
Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture.
- October 2007
Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.
Authors: Gravotta D, Ami Deora, Perret E, Oyanadel C, Soza A, Ryan Schreiner, Gonzalez A, Enrique Rodriguez-Boulan
- Proceedings of the National Academy of Sciences
- January 30, 2007
The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.
- Current Opinions in Cell Biology
- August 2005
The cell biologist's insight into endosomal diversity, in terms of both form and function, has increased dramatically in the past few years. This understanding has been promoted by the availability of powerful new techniques that allow imaging of both cargo and machinery in the endocytic process in real time, and by our ability to inhibit components of this machinery by RNA interference. The emerging picture from these studies is of a highly complex, dynamic and adaptable endosomal system that interacts at various points with the secretory system of the cell.
The Rfb Genes in Azotobacter Vinelandii Are Arranged in a Rfbfgc Gene Cluster: A Significant Deviation to the Arrangement of the Rfb Genes in Enterobacteriaceae.
- Biochemical and Biophysical Research Communications
- April 17, 1998
We report the identification of rfbF and rfbC located adjacent to the previously identified rfbG (Gavini et. al. Biochem. Biophys. Res. Commun. 1997, 240, 153-161) from the non-symbiotic, non-pathogenic soil bacterium Azotobacter vinelandii. The rfbF open reading frame encodes a putative polypeptide of 256 amino acids. This polypeptide shares a homology of 74% with the RfbF of Synechocystis sp. and a 70% homology with the AscA of Yersinia pseudotuberculosis which function as alpha-D-glucose-1-phosphate cytidylyltransferases in the biosynthesis of the O-antigen. The rfbC encodes a putative polypeptide of 186 amino acids. It shows strongest homology to the RfbC of Synechocystis sp. (64%) and Salmonella typhimurium (40%). RfbC functions as a dTDP-4-Dehydrorhamnose 3,5-Epimerase. The genes identified here have a low G + C content (approximately 56%) as compared to the A. vinelandii chromosome (approximately 63%) which is characteristic of the rfb clusters identified in other bacteria and may be indicative of the acquisition of the rfb genes by interspecific gene transfer. Despite the high level of sequence conservation, the organization of the rfb genes in A. vinelandii deviates from the arrangement of the most thoroughly studied rfb gene clusters of Enterobacteriaceae.
Identification and Mutational Analysis of Rfbg, the Gene Encoding CDP-D-Glucose-4,6-Dehydratase, Isolated from Free Living Soil Bacterium Azotobacter Vinelandii.
- Biochemical and Biophysical Research Communications
- November 7, 1997
We have identified the rfbG from a non-symbiotic and non-pathogenic soil bacterium, Azotobacter vinelandii. The nucleotide sequence analysis of the rfbG revealed an open reading frame that encodes a peptide of 360 amino acids. This deduced peptide shares 57% homology with the RfbG of Synechocystis and 47% homology with the RfbG of Yersinia pseudotuberculosis. The previously identified short-chain dehydrogenases/reductases family signature sequence is conserved in the sequence of the RfbG of A. vinelandii. Southern blotting analysis of A. vinelandii chromosome by probed with 1.1 kb PstI DNA fragment corresponding to rfbG revealed that it is present as single copy on A. vinelandii chromosome. Disrupting the rfbG present on the chromosome of A. vinelandii, by insertion of kanamycin resistance marker via homologous recombination, resulted in drastic changes in the growth characteristics. The rfbG-negative A. vinelandii grown in liquid medium exhibited agglutination that is characteristic of rfb- mutants of other bacteria, suggesting that we have cloned the functional copy of the rfbG of A. vinelandii.
Ryan Schreiner's Skills & Expertise
- Molecular Biology
- Cell Biology
- Cell Culture
- Tissue Culture
- Life Sciences
- Western Blotting
- Flow Cytometry
- Fluorescence Microscopy
- Protein Chemistry
- Confocal Microscopy
- Molecular Cloning
- Protein Expression
- Protein Purification
- Assay Development
- In Vitro
- In Vivo
- RNA isolation
- Animal Models
- Stem Cells
- Cell Signaling