MODULATION OF ENDOCYTIC AND EXOCYTIC TRAFFIC IN POLARIZED EPITHELIAL CELLS

Efficient and selective delivery and retrieval of cargo to polarized plasma membrane domains are essential for the maintenance of epithelial cell function. My studies focused on events involved in protein endocytosis from and delivery to the apical surface. Endocytosis is a multi-step process that is crucial for cells to take up nutrients and modulate receptor density. Temporal synthesis and degradation of the lipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] is critical for endocytosis to proceed. PtdIns(4,5)P2 is generated by three differentially localized phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) isoforms (α, β, and γ). My studies examined the requirement for PtdIns(4,5)P2 in apical and basolateral endocytosis in polarized kidney epithelial cells by testing the effects of overexpressing individual PIP5KI isoforms. I found that PtdIns(4,5)P2 synthesis by PIP5KIβ is rate limiting for apical but not basolateral endocytosis, and that this is likely due to changes in coated pit maturation at the apical plasma membrane. The selective modulation of apical endocytosis by PtdIns(4,5)P2 has potential consequences on the maintenance of kidney cell function. Maintaining apical and basolateral membrane domain composition requires selective vesicle fusion and is mediated by Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor (SNARE) proteins. A number of pathways exist for the delivery of newly synthesized proteins to these surface domains and the post-Golgi vesicle SNAREs, or VAMPs, that mediate fusion in these pathways have not been identified. I sought to determine the role of VAMP7 in these pathways. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and changes in VAMP7 levels modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested but did decrease the length and frequency of primary cilia and disrupted cyst morphology. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in a MDCK lysosomal storage disorder model. Together, my data suggest that VAMP7 plays an essential role in ciliogenesis and cystogenesis. This is the first study implicating an R-SNARE in ciliogenesis and cystogenesis

Two-Phase Flow Experiments on Counter-Current Flow Limitation in a model of the Hot Leg of a Pressurized Water Reactor (2015 test series): Two-Phase Flow Experiments on Counter-Current Flow Limitation in a model of the Hot Leg of a Pressurized Water Reactor (2015 test series)

Counter-Current Flow Limitation (CCFL) is of importance for PWR safety analyses in several accident scenarios connected with loss of coolant. Basing on the experiences obtained during a first series of hot leg tests now new experiments on counter-current flow limitation were conducted in the TOPFLOW pressure vessel. The test series comprises air-water tests at 1 and 2 bar as well as steam-water tests at 10, 25 and 50 bar. During the experiments the flow structure was observed along the hot leg model using a high-speed camera and web-cams. In addition pressure was measured at several positions along the horizontal part and the water levels in the reactor-simulator and steam-generator-simulator tanks were determined. This report documents the experimental setup including the description of operational and special measuring techniques, the experimental procedure and the data obtained. From these data flooding curves were obtained basing on the Wallis parameter. The results show a slight shift of the curves in dependency of the pressure. In addition a slight decrease of the slope was found with increasing pressure. Additional investigations concern the effects of hysteresis and the frequencies of liquid slugs. The latter ones show a dependency on pressure and the mass flow rate of the injected water. The data are available for CFD-model development and validation

Persistence of frequency in gas–liquid flows across a change in pipe diameter or orientation

From a study of the characteristics of structures across a 67/38 mm sudden contraction, using air/silicone oil flows, it has been found that frequencies of the structures (mainly slugs) persist across the contraction. This is in contrast to the velocities and lengths which increase as they move into the smaller diameter pipe. These observations were found for both vertical and 5° upward orientations. A similar persistence of frequency has been found from four other sources in the literature: a vertical (gradual) contraction; a horizontal Venturi; and two cases of horizontal pipe, 90° bend and vertical riser combination. The latter were at two contrasting conditions: (i) at atmospheric pressure with air/water in small diameter (34 mm) pipes; (ii) at 20 bar in larger diameter pipes (189 mm) using nitrogen and naphtha

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

VAMP7 modulates ciliary biogenesis in kidney cells

Epithelial cells elaborate specialized domains that have distinct protein and lipid compositions, including the apical and basolateral surfaces and primary cilia. Maintaining the identity of these domains is required for proper cell function, and requires the efficient and selective SNARE-mediated fusion of vesicles containing newly synthesized and recycling proteins with the proper target membrane. Multiple pathways exist to deliver newly synthesized proteins to the apical surface of kidney cells, and the post-Golgi SNAREs, or VAMPs, involved in these distinct pathways have not been identified. VAMP7 has been implicated in apical protein delivery in other cell types, and we hypothesized that this SNARE would have differential effects on the trafficking of apical proteins known to take distinct routes to the apical surface in kidney cells. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and siRNA-mediated knockdown modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested, but did decrease the length and frequency of primary cilia. Additionally, VAMP7 knockdown disrupted cystogenesis in cells grown in a three-dimensional basement membrane matrix. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in an MDCK lysosomal storage disorder model. Together, our data suggest that VAMP7 plays an essential role in ciliogenesis and lumen formation. To our knowledge, this is the first study implicating an R-SNARE in ciliogenesis and cystogenesis. © 2014 Szalinski et al

Structures in gas–liquid churn flow in a large diameter vertical pipe

Gas–Liquid two phase co-current flow in a vertical riser with an internal diameter of 127 mm was investigated in the churn flow pattern. This paper presents detailed experimental data obtained using a Wire Mesh Sensor. It shows that the most obvious features of the flow are huge waves travelling on the liquid film. Wisps, large tendrils of liquid and the product of incomplete atomisation, which had previously detected in smaller diameter pipes, have also been found in the larger diameter pipe employed here. The output of the Wire Mesh Sensor has been used to determine the overall void fraction. When examined within a drift flux framework, it shows a distribution coefficient of ∼1, in contrast to data for lower gas flow rates. Film thickness time series extracted from the Wire Mesh Sensor output have been examined and the trends of mean film thickness, that of the base film and the wave peaks are presented and discussed. The occurrence of wisps and their frequencies have been quantified