ATPase activity, proton gradients and proton secretion inhibition during experiments with Sepia officinalis and Sepioteuthis lessoniana

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco2 between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE3) and Na+-K+-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

Polar membrane lipids and elemental composition of Thermococcus kodakarensis cells according to growth stage and phosphorus supply

We observed significant changes in the elemental and intact polar lipid (IPL) composition of the archaeon Thermococcus kodakarensis (KOD1) in response to growth stage and phosphorus supply. Reducing the amount of organic supplements and phosphate in growth media resulted in significant decreases in cell size and cellular quotas of carbon (C), nitrogen (N), and phosphorus (P), which coincided with significant increases in cellular IPL quota and IPLs comprising multiple P atoms and hexose moieties. Relatively more cellular P was stored as IPLs in P-limited cells (2-8%) compared to control cells (<0.8%). We also identified a specific IPL biomarker containing a phosphatidyl-N-acetylhexoseamine headgroup that was relatively enriched during rapid cell division. These observations serve as empirical evidence of IPL adaptations in Archaea that will help to interpret the distribution of these biomarkers in natural systems. The reported cell quotas of C, N, and P represent the first such data for a specific archaeon and suggest that thermophiles are C-rich compared to the cell carbon-to-volume relationship reported for planktonic bacteria.

In-situ and Ex-situ characterization of III-V semiconductor materials and solar cells upon 10 MEV proton irradiation

In this work we present the results and analysis of a 10 MeV proton irradiation experiment performed on III-V semiconductor materials and solar cells. A set of representative devices including lattice-matched InGaP/GaInAs/Ge triple junction solar cells and single junction GaAs and InGaP component solar cells and a Ge diode were irradiated for different doses. The devices were studied in-situ before and after each exposure at dark and 1 sun AM0 illumination conditions, using a solar simulator connected to the irradiation chamber through a borosilicate glass window. Ex-situ characterization techniques included dark and 1 sun AM0 illumination I-V measurements. Furthermore, numerical simulation of the devices using D-AMPS-1D code together with calculations based on the TRIM software were performed in order to gain physical insight on the experimental results. The experiment also included the proton irradiation of an unprocessed Ge solar cell structure as well as the irradiation of a bare Ge(100) substrate. Ex-situ material characterization, after radioactive deactivation of the samples, includes Raman spectroscopy and spectral reflectivity.

Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors

The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near −50 mV and −200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 μM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5–10 s and relaxed over 40–60 s thereafter. The [Ca2+]i increases showed a voltage threshold near −120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in “conditioning” [Ca2+]i signals that regulate ion channels for stomatal function.

Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value (≈1,200 μM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin

NMR investigations have been carried out of complexes between bovine chymotrypsin Aα and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-l-Phe-CF3, N-Acetyl-Gly-l-Phe-CF3, N-Acetyl-l-Val-l-Phe-CF3, and N-Acetyl-l-Leu-l-Phe-CF3. The D/H fractionation factors (φ) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hδ1) in these four complexes at 5°C were in the range φ = 0.32–0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hɛ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 8.97–9.18), the exchange rate of the His 57-Hδ1 proton with bulk water protons (284–12.4 s−1), and the activation enthalpies for this hydrogen exchange (14.7–19.4 kcal⋅mol−1). It was found that the previously noted correlations between the inhibition constants (Ki 170–1.2 μM) and the chemical shifts of His 57-Hδ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 18.61–18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940–11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hδ1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements.

Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.