INF2 mutations appear to cause many cases of FSGS-associated Charcot-Marie-Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function. (Funded by the Agence Nationale de la Recherche and others.).

We give an exact self-consistent operator description of the spin and orbital angular momenta, position, and spin-orbit interactions of nonparaxial light in free space. Both quantum-operator formalism and classical energy-flow approach are presented. We apply the general theory to symmetric and asymmetric Bessel beams exhibiting spin-and orbital-dependent intensity profiles. The exact wave solutions are clearly interpreted in terms of the Berry phases, quantization of caustics, and Hall effects of light, which can be readily observed experimentally.

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

No abstract

The assembly of signaling nanoterritories at the T cell immunological synapse is controlled by the coordinated trafficking and fusion of specific vesicles containing the signaling molecules Lck, LAT, and TCRζ.

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.

We have isolated a human cDNA that is expressed in the intermediate and late stages of T-cell differentiation. The cDNA encodes a highly hydrophobic protein, termed MAL, that lacks a hydrophobic leader peptide sequence and contains four potential transmembrane domains separated by short hydrophilic segments. The predicted configuration of the MAL protein resembles the structure of integral proteins that form pores or channels in the plasma membrane and that are believed to act as transporters of water-soluble molecules and ions across the lipid bilayer. The presence of MAL mRNA in a panel of T-cell Total cytoplasmic RNA from tissue culture cells was prepared by the Nonidet P-40 lysis method (6). Membranebound RNA was prepared by mechanical disruption of cells in hypotonic buffer and differential centrifugation (7). When frozen tissues were used, total RNA was isolated by homogenization in 4 M guanidinium thiocyanate, followed by ultracentrifugation through a 5.7 M CsCl cushion (8). Poly-(A)' RNA was isolated by oligo(dT)-cellulose chromatography (9). High molecular weight genomic DNA was prepared essentially as described by Maniatis et al. (10).The first strand of cDNA was synthesized by oligo(dT) priming using poly(A)+ RNA from MOLT-4 cells and avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Petersburg, FL) in the presence of actinomycin D at 100 ng/ml. This cDNA was mixed with a 10-fold mass excess of poly(A)+ RNA from CCRF HSB-2 cells, boiled for 60 sec, and incubated at 680C in 0.5 M phosphate buffer, pH 6.8/5 mM EDTA/0. 1% NaDodSO4 to a Rot (initial concentration of RNA x time) value of 1500 (11). Unhybridized cDNA was separated from the cDNA RNA hybrids by chromatography on a hydroxyapatite column using 0.12 M phosphate buffer, pH 6.8/0.1% NaDodSO4 at 60°C. This single-stranded cDNA fraction was then used to construct libraries in the EcoRI site of pBR322 (10) and bacteriophage XgtlO (12). The second strand was synthesized by using RNase H, Escherichia coli DNA polymerase, and T4 DNA ligase (13). cDNA molecules >800 base pairs long were cloned in the unique EcoRI site of XgtlO. Subtracted [32P]cDNA probes were generated using a similar protocol except that the cDNA was labeled to specific activities of up to 109 cpm/,ug (11). Screening was carried out using 106 cpm per 137-mm filter under standard conditions (10).High molecular weight genomic DNA was digested with restriction endonucleases and blotted as described by Southern (14). For RNA blots, RNA was denatured in the presence of 50% (vol/vol) formamide and 2.2 M formaldehyde, subjected to electrophoresis on 1.2% agarose/formaldehyde gels, and blotted as described by Thomas (15). Final blot washing conditions were 0.1x SSC/0.1% NaDodSO4 at 500C. (lx SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0.)Restriction fragments from XMA5 and pMA34 inserts were subcloned into the M13mp8 vector and sequenced (16).The full-length cDNA insert from XMA5 was subcloned in the appropriate orientation in the EcoRI site of pSP65 (17). Trans...

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.