Cross-linking Of Membrane Proteins Research Articles

Thf1 interacts with PS I and stabilizes the PS I complex in Synechococcus sp. PCC7942.

Thylakoid formation1 protein (Thf1) is a multifunctional protein that is conserved in all photosynthetic organisms. In this study, we used the model cyanobacterium Synechococcus sp. PCC7942 (hereafter Synechococcus) to show that the level of Thf1 is altered in response to various stress conditions. Although this protein has been reported to be involved in thylakoid formation, the thylakoid membrane in the thf1 deletion strain (ΔThf1) was not affected. Compared with the WT, ΔThf1 showed reduced PS II activity, with increased levels of D1 under high light (HL) conditions, which was resulted from blocked D1 degradation by the FtsH protease and thus inhibits PS II repair. PS I was found to be more seriously affected than PS II in ΔThf1, even under low light conditions, suggesting that PS I damage could be the primary effect of thf1 deletion in Synechococcus. Further analysis revealed that the ΔThf1 mutant had a lower PS I subunit content and lower PS I stability under HL conditions. Further sucrose gradient fractionation of the membrane protein complexes and crosslinking and immunoblot analysis indicated that Thf1 interacts with PS I. Together, our results reveal that Thf1 interacts with PS I and thereby stabilizes PS I in Synechococcus.

Further evidence for a membrane receptor that binds glucocorticoids in the rodent hypothalamus

In parallel with their well-characterized delayed genomic effects, steroid hormones exhibit rapid, non-genomic effects at molecular, cellular and behavioral levels. We have proposed a model of rapid, non-genomic glucocorticoid inhibition of hypothalamic neuroendocrine cells through a putative membrane-associated glucocorticoid receptor (GR). Here we tested for plasma membrane GR immunoreactivity and binding in the hypothalamic supraoptic and paraventricular nuclei. Selective cross-linking of membrane proteins with membrane-impermeant BS3 and subsequent Western blot analysis with a monoclonal GR antibody revealed a reduction in the intensities of a ∼98kDa immunoreactive band and a ∼64kDa band in the rat paraventricular and supraoptic nuclei, and of a 64kDa band in hippocampal tissue, which suggested that these proteins are associated with the membrane. Saturation binding of [3H]-corticosterone and [3H]-dexamethasone in rat and mouse hypothalamic tissue revealed a Kd 4–24-fold lower and a Bmax 4–7-fold lower for the membrane-associated GR compared to the intracellular GR, suggesting a lower affinity and abundance of the glucocorticoid binding sites in the membrane than in the cytosol. Together, these findings suggest the presence of a low-affinity, low-abundance membrane-associated GR in the hypothalamus that shares homology with the intracellular GR, and are consistent with physiological evidence of rapid, non-genomic glucocorticoid actions in hypothalamic neuroendocrine cells that are GR dependent.

Nonlinear laser scanning microscopy of oral multispecies-biofilms: fixative induced fluorescence as a fast and economical in vitro screening method

Abstract In this letter we report a fast and easy method which could be used for initial screening of multispecies-biofilm development on putative new dental implant materials. Most staining methods require numerous washing steps that can result in detachment of loosely bound biofilms and therefore falsify the results. Thus, we used glutaraldehyde fixation, which induces autofluorescence through bacterial membrane protein cross-linking and concurrently stabilizes the biofilm structure. We analyzed the biofilms with nonlinear laser scanning microscopy and were able to (I) evaluate the multispecies-biofilm growth and (II) distinguish between bacterial species based on different two-photon autofluorescence intensities.

Microdomain Formation Controls Spatiotemporal Dynamics of Cell-Surface Glycoproteins.

The effect of galectin-mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC-1) was studied qualitatively and quantitatively by high-resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid-bearing proteins (SABPs) and mucin-type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne-conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin-1 or -3. Strong effects of galectin-mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin-mediated crosslinking was accurately mimicked by streptavidin-mediated crosslinking of biotin-tagged glycoproteins and verified by single-molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.

Overexpression of Tumor Necrosis Factor-α in the Lungs Alters Immune Response, Matrix Remodeling, and Repair and Maintenance Pathways

Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis.

Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins

Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS–PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.

Anaerobic storage of red blood cells.

Anaerobic storage of red blood cells.

Factor XIII deficiency

Inherited factor XIII (FXIII) deficiency is a rare bleeding disorder that can present with umbilical bleeding during the neonatal period, delayed soft tissue bruising, mucosal bleeding and life-threatening intracranial haemorrhage. FXIII deficiency has also been associated with poor wound healing and recurrent miscarriages. FXIII plays an integral role in haemostasis by catalysing the cross-linking of fibrin, platelet membrane and matrix proteins throughout thrombus formation, thus stabilizing the blood clot. The molecular basis of FXIII deficiency is characterized by a high degree of heterogeneity, which contributes to the different clinical manifestations of the disease. There have been more than 60 FXIII mutations identified in the current literature. In addition, single nucleotide polymorphisms have been described, some of which have been shown to affect FXIII activity, contributing further to the heterogeneity in patient presentation and severity of clinical symptoms. Although there is a lifelong risk of bleeding, the prognosis is excellent when current prophylactic treatment is available using cryoprecipitate or plasma-derived FXIII concentrate.

Over‐expression of GTP‐binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler’s murine encephalomyelitis virus infection

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus-induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over-expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP-binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV-infected astrocytes, peaking 2-4 h after infection. Over-expression is also induced by the inflammation-related chemokines interleukin-6 and interferon-gamma but not by interleukin-1alpha or tumor necrosis factor-alpha. From the many GTPases that could be over-expressed we have studied two, because of its biological significance; Ras p21 and the subunit alphai2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione-S-transferase-fusion protein, following TMEV infection. A final demonstration of physiological up-regulation is provided by UV cross-linking of membrane proteins with the hydrolysis-resistant GTP agonist GTP [gamma-(35)S]. This technique allow us to detect, after SDS-PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Galphai2 subunit of protein G, Munc-18 and protein interacting with C kinase 1, was performed by real-time RT-PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT-PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up-regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.

On the Opening of an Insensitive Cyclosporin A Non-specific Pore by Phenylarsine Plus Mersalyl

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.

Human Organic Anion Transporter hOAT1 Forms Homooligomers

Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.

Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter

The Na +-Cl −-K + isoform 1 (NKCC1) is a co-transporter that increases the intracellular concentration of chloride. NKCC1 plays a critical role in neuronal excitability and it has been recently suggested that it can contribute to hyperalgesic states by modulating the chloride concentration inside nociceptive neurons. In the spinal cord, trafficking of neurotransmitter receptors from the cytosol to the plasma membrane has been demonstrated to contribute to the development of hyperalgesia. However, it is unknown if trafficking of co-transporters can also occur in the nervous system or if it can be induced by painful stimulation. In this study, we have induced referred mechanical hyperalgesia in vivo by intracolonic instillation of capsaicin in mice. Using subcellular fractionation of proteins and cross-linking of membrane proteins we have observed that intracolonic capsaicin induced a 50% increase in NKCC1 in the plasma membrane of lumbosacral spinal cord 90 and 180 min after instillation, in parallel with a similar decrease in the cytosolic fraction. These effects returned to basal levels 6 h after capsaicin treatment. Intracolonic capsaicin also evoked a rapid (10 min) and transient phosphorylation of NKCC1, however, intracolonic saline did not produce significant changes in either NKCC1 trafficking or phosphorylation and none of the treatments induced any alterations of NKCC1 in the thoracic spinal cord. These results suggest that phosphorylation and recruitment of NKCC1 might play a role in referred mechanical hyperalgesia evoked by a painful visceral stimulus. The time course of the effects observed suggests that phosphorylation could contribute to the initial generation of hyperalgesia whereas trafficking could participate in the maintenance of hyperalgesic states observed at longer time points.

Role of Mechanical Stress on the Transglutaminase -Mediated Crosslinking of Erythrocyte Membrane Proteins.

The life-span of erythrocytes is relatively constant within species and it may be determine by the number of times the cell passes through the circulatory system — enduring repeated cycles of mechanical stress. The intrinsic transglutaminase is the major enzyme capable of catalyzing covalent γ-glutamyl-ε-lysine crosslinks that enhance erythrocyte membrane rigidity. Here we report the cloning of mouse erythrocyte transglutaminase (TG) and the role of mechanical stress on the TG-mediated crosslinking of erythrocyte membrane proteins. TG was PCR cloned from reticulocyte cDNA by using consensus primers. Northern blot analysis of the polyA+ mRNA revealed the TG transcript to be 4.6 kb. Recombinant TG was expressed in yeast and the enzymatic crosslinking was detected by labeling glutamine (Q) acyl-donors in inside out vesicles (IOVs) with fluorescent dansylcadaverine (DNC), which serves as the acyl-acceptor. Crosslinking of spectrin/ankyrin, protein 4.1, and band 3 regions (known Q-donors) by TG required CaCl2 at 0.1–0.25 mM and was regulated by both GTP (optimal at 0.01–0.05 mM) and ATP (optimal at 0.5–2.0 mM) with inhibitory effects at higher concentrations. Protein 4.2 (P4.2), a TG pseudo-enzyme, and the cytoplasmic domain of band 3 (cdb3) were also produced recombinantly which allowed for an in-vitro model to test molecular interactions with TG. Specifically, the crosslinking of cdb3 by TG and the binding of P4.2 and TG to cdb3 were examined. A rise in [CaCl2] to near 1 mM showed increased activity of TG (~75% of maximal activity) together with a slight decrease in TG binding affinity to cdb3 (~25% decrease). The crosslinking of cdb3 by TG was inhibited by P4.2, however the presence of P4.2 facilitated the binding of TG to cdb3. The analysis of endogenous TG activity in intact erythrocytes subjected to mechanical stress by hypo-osmolarity (equi-biaxial stretch) or shear stress (anisotropic extension) was also performed. Hypo-osmotically stressed DNC-loaded erythrocytes in the presence of 2 mM [CaCl2] produced a profile with a less intense crosslinking as compared to that of IOVs. In addition, two novel Q-donors (Q1 and Q2) that required mechanical deformation of the membrane to be crosslinked were discovered. Mechanical shearing of erythrocytes in a viscometer produced a similar profile, again with Q2 being the most crosslinked Q-donor. The ratio of Q2/band 3 crosslinking under shear, however, was two fold greater than that under hypo-osmotically induced stress, indicating that varying degrees of crosslinking may be induced by different modes of mechanical stress. Interestingly, Q2 also became crosslinked in IOVs prepared from erythrocytes previously hyposmotically incubated in the presence of extracellular calcium. The importance of mechanical deformation and calcium influx in the binding of Q2 to membrane-associated proteins was further supported by the findings that calcium introduced into erythrocytes using ionophore A23187 alone was not sufficient to induce the crosslinking of Q2. Together these experiments support our hypothesis that periodic mechanical stress may serve as an inherent molecular timer. It is likely that the activation of TG by transient increases in near-membrane calcium concentrations may lead to the accumulation of crosslinks of proteins at the membrane and an eventual entrapment of less deformable erythrocytes in the sinusoids in the spleen followed by subsequent phagocytosis through the recognition of band 3 auto-antibodies.

Activation of transglutaminase in μ-calpain null erythrocytes

Intracellular transglutaminases (protein–glutamine: amine γ-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, μ-calpain. Using μ-calpain null ( Capn1 −/−) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of μ-calpain. Also, the absence of μ-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of μ-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.

Protein disulfide isomerase, a component of the estrogen receptor complex, is associated with Chlamydia trachomatis serovar E attached to human endometrial epithelial cells.

Chlamydia trachomatis serovar E, the leading bacterial agent responsible for sexually transmitted diseases, is required to invade genital epithelial cells for its growth and survival, yet little is known about the adhesin-receptor interactions promoting its entry. In contrast, much has been published on the heparan sulfate receptor for binding C. trachomatis L2 elementary bodies (EBs) prior to entry into HeLa cells. Using a different experimental approach in which a biotinylated apical membrane protein receptor(s) attached to EB at 4 degrees C was stripped off the surface of polarized HEC-1B cells and immunoprecipitated with polyclonal anti-EB antibodies, an approximately 55-kDa protein was reproducibly detected by enhanced chemiluminescence and two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization mass-spectrometry sequence analysis revealed the 55-kDa protein to be protein disulfide isomerase (PDI), a member of the estrogen receptor complex which carries out thiol-disulfide exchange reactions at infected host cell surfaces. Exposure of HEC-1B cells during EB attachment (1.5 to 2 h) to three different inhibitors of PDI reductive reactions--(i) the thiol-alkylating reagent DTNB (5,5'-dithiobis[2-nitrobenzoic acid]), (ii) bacitracin, and (iii) anti-PDI antibodies--resulted in reduced chlamydial infectivity. Since (i) C. trachomatis serovar E attachment to estrogen-dominant primary human endometrial epithelial cells is dramatically enhanced and (ii) productive entry into and infectivity of EB in host cells is dependent on reduction of EB cross-linked outer membrane proteins at the host cell surface, these data provide some preliminary evidence for an intriguing new potential receptor candidate for further analysis of luminal C. trachomatis serovar E entry.

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like α-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.

Antioxidant activity of the organotellurium compound 3-[4-( N, N-dimethylamino)benzenetellurenyl]propanesulfonic acid against oxidative stress in synaptosomal membrane systems and neuronal cultures

Antioxidant activities of 3-[4-( N, N-dimethylamino) benzenetellurenyl]propanesulfonic acid sodium salt (NDBT) were evaluated in solution, red blood cells, synaptosomal membranes, and cultured hippocampal neuronal cells after exposure to peroxynitrite (ONOO −) and hydroxyl radicals. The organotellurium compound NDBT possesses significant activity towards hydrogen peroxide and/or the hydroxyl radical in solution, demonstrated by inhibition of hydroxylation of terephthalic acid. In addition, the compound displayed great antioxidant abilities as shown by: reduction of ONOO −-induced 2,7-dichlorofluorescein (DCF) fluorescence in synaptosomes; complete prevention of lipid peroxidation in synaptosomes caused by OH radicals (TBARS), and significant prevention of protein oxidation caused by ONOO − and OH, indexed by the levels of protein carbonyls in synaptosomes and neuronal cells. The presence of the compound abolished neuronal cell death caused by ONOO −. Further, the compound was effective in preventing the oxidative changes in synaptosomal membrane protein conformation and crosslinking (EPR spin labeling). Finally, the organotellurium molecule attenuated peroxynitrite-induced, luminol-dependent chemiluminescence in red blood cells — an index of cellular oxidation. These findings demonstrate the great potential of the antioxidant and are consistent with the notion that NDBT may have a role to play in modulating oxidative stress in neurodegenerative disorders, including Alzheimer’s disease.

Dopamine induced protein damage in mitochondrial-synaptosomal fraction of rat brain

Dopamine during in vitro oxidation induced covalent cross-linking of membrane proteins in rat brain crude mitochondrial-synaptosomal fraction. The process is not inhibited by hydroxyl radical scavengers, lipid soluble anti-oxidants, metal-chelator or catalase, but reduced glutathione produced a dramatic inhibition of cross-linking. The protein cross-linking mediated by dopamine is not associated with any detectable membrane lipid peroxidation but significant formation of protein bound quinone takes place during incubation. Our results indicate that reactive quinones rather than oxygen free radicals are involved in dopamine induced protein cross-linking in rat brain membrane fraction.

The Nudel Protease of Drosophila Is Required for Eggshell Biogenesis in Addition to Embryonic Patterning

The dorsoventral axis of the Drosophila embryo is defined by a ventral signal that arises within the perivitelline space, an extracellular compartment between the embryo plasma membrane and the vitelline membrane layer of the eggshell. Production of the ventral signal requires four members of the serine protease family, including a large modular protein with a protease domain encoded by the nudel gene. Here we provide evidence that the Nudel protease has an integral role in eggshell biogenesis. Mutations in nudel that disrupt Nudel protease function produce eggs having vitelline membranes that are abnormally permeable to the dye neutral red. Permeability varies among mutant nudel alleles but correlates with levels of Nudel protease catalytic activity and function in embryonic dorsoventral patterning. These mutations also block cross-linking of vitelline membrane proteins that normally occurs upon egg activation, just prior to fertilization. In addition, Nudel protease autoactivation temporally coincides with vitelline membrane cross-linking and can be triggered in mature eggs in vitro by conditions that lead to egg activation. We discuss how the Nudel protease might be involved in both eggshell biogenesis and embryonic patterning.

Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells.

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.