Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Proteolytic mechanisms of cell injury following glucose -oxygen deprivation in primary neuronal cultures

Researchers have historically emphasized the contribution of caspase-3 to apoptotic but not necrotic cell death, while calpain has been implicated primarily in necrosis and, to a lesser extent, in apoptosis. Activation of these proteases occurs in vivo following various CNS insults including ischemia. In addition, both necrotic and apoptotic cell death phenotypes are detected following ischemia. However, the contributions of calpain and caspase-3 to apoptotic and necrotic cell death phenotypes following CNS insults are relatively unexplored. To date, no study has examined the concurrent activation of calpain and caspase-3 in necrotic and apoptotic cell death phenotypes following any CNS insult. The present study employed oxygen-glucose deprivation (OGD) to determine the relative contributions of caspase-3 and calpain to apoptotic and necrotic cell death following OGD. Experiments characterized a model of OGD by evaluating cell viability and characterizing the cell death phenotypes following OGD in primary septo-hippocampal co-cultures. Furthermore, cell markers (NeuN and MAP2 or GFAP) assessed the effects of OGD on neuronal and astroglial viability, respectively. In addition, calpain and caspase-3 mediated proteolysis of α-spectrin was examined using Western blot techniques. Activation of these proteases in individual cells phenotypically characterized as apoptotic and necrotic was also evaluated by using antibodies specific for calpain or caspase-3 mediated breakdown products to α-spectrin. Administration of appropriate caspase-3 and calpain inhibitors also examined the effects of protease inhibition on cell death. OGD produced prominent expression of apoptotic cell death phenotypes primarily in neurons, with relatively little damage to astroglia. Although Western blot data suggested greater proteolysis of α-spectrin by calpain than caspase-3, co-activation of both proteases was usually detected in cells exhibiting apoptotic or necrotic cell death phenotypes. While inhibition of calpain and caspase-3 activity decreased LDH release following OGD, it was not clear whether this effect was also associated with a decrease in cell death and the appearance of apoptotic cell death phenotypes. These data demonstrate that both calpain and caspase-3 contribute to the expression of apoptotic cell death phenotypes following OGD, and that calpain could potentially have a larger role in the expression of apoptotic cell death than previously thought. ^

The role of Ski/Sno oncoproteins as negative regulators of the TGF-beta/SMAD signaling pathway in B -cell non -Hodgkin's lymphoma (NHL-B)

Non-Hodgkin's lymphomas are common tumors of the human immune system, primarily of B cell lineage (NHL-B). Negative growth regulation in the B cell lineage is mediated primarily through the TGF-β/SMAD signaling pathway that regulates a variety of tumor suppressor genes. Ski was originally identified as a transforming oncoprotein, whereas SnoN is an isoform of the Sno protein that shares a large region of homology with Ski. In this study, we show that Ski/SnoN are endogenously over-expressed both in patients' lymphoma cells and NHL-B cell lines. Exogenous TGF-β1 treatment induces down-regulation of Ski and SnoN oncoprotein expression in an NHL-B cell line, implying that Ski and SnoN modulate the TGF-β signaling pathway and are involved in cell growth regulation. Furthermore, we have developed an NHL-B cell line (DB) that has a null mutation in TGF-β receptor type II. In this mutant cell line, Ski/SnoN proteins are not down-regulated in response to TGF-β1 treatment, suggesting that downregulation of Ski and SnoN proteins in NHL-B require an intact functional TGF-β signaling pathway Resting normal B cells do not express Ski until activated by antigens and exogenous cytokines, whereas a low level of SnoN is also present in peripheral blood Go B cells. In contrast, autonomously growing NHL-B cells over-express Ski and SnoN, implying that Ski and SnoN are important cell cycle regulators. To further investigate a possible link between reduction of the Ski protein level and growth inhibition, Ski antisense oligodeoxynucleotides were transfected into NHL-B cells. The Ski protein level was found to decrease to less than 40%, resulting in restoring the effect of TGF-β and leading to cell growth inhibition and G1 cell cycle arrest. Co-immunoprecipitation experiments demonstrated that Ski associates with Smad4 in the nucleus, strongly suggesting that over-expression of the nuclear protein Ski and/or SnoN negatively regulates the TGF-β pathway, possibly by modulating Smad-mediated tumor suppressor gene expression. Together, in NHL-B, the TGF-β/SMAD growth inhibitory pathway is usually intact, but over-expression of the Ski and/or SnoN, which binds to Smad4, abrogates the negative regulatory effects of TGF-β/SMAD in lymphoma cell growth and potentiates the growth potential of neoplastic B cells. ^

Endogenous nitric oxide regulates p53 signaling and DNA damage-induced apoptosis in melanoma cells

Nitric oxide is involved in a multitude of processes including regulation of vascular tone, neurotransmission, immunity, and cancer. Evidence suggests that nitric oxide exhibits anti-apoptotic activity in melanoma cells. Our laboratory showed that tumor expression of inducible nitric oxide synthase correlated strongly with poor survival in stage III and IV melanoma patients, suggesting an antagonistic role for nitric oxide in melanoma response to therapy. Therefore, the hypothesis that endogenously produced nitric oxide antagonizes chemotherapy-induced apoptosis was formed. Using cisplatin as a model for DNA damage in melanoma cell lines, the capacity of nitric oxide to regulate cell growth and apoptotic responses to cisplatin treatment was examined. The depletion of endogenously generated nitric oxide resulted in changes in cell cycle regulation and enhanced cisplatin-induced apoptosis in melanoma cells. Since nitric oxide was shown to be involved in the regulation of p53 stability, conformation and DNA binding activity, whether signaling through wild-type p53 in melanoma cells is regulated by nitric oxide was tested. Cisplatin-induced p53 accumulation and p21Waf1/Cip1/Sdi1 expression in nitric oxide-depleted melanoma cells were found to be strongly suppressed. When p53 binding to the p21Waf1/Cip1/Sdi1 promoter was examined, it was found that nitric oxide depletion significantly reduced the cisplatin-induced formation of p53-DNA complexes. These results suggest that nitric oxide is required for activation of wild-type p53 after DNA damage in melanoma cells. Finally, whether signaling through p53 controls melanoma response to DNA damage was examined. Transfection of a plasmid containing a dominant negative form of mutated p53 inhibited p21 Waf1/Cip1/Sdi1 expression and concomitantly enhanced apoptosis after cisplatin treatment. These data suggest that the induction of wild-type p53 protects melanoma cells against DNA damage via the up-regulation of p21 Waf1/Cip1/Sdi1. Together, these data strongly support the model that endogenous nitric oxide is required for p53 activation and p21Waf1/Cip1/Sdi1 expression after DNA damage, which can enhance melanoma resistance to therapy. Thus, in context of melanoma cells with wild-type p53 , low levels of endogenous constitutively-produced nitric oxide appear to facilitate the activation of p53 in response to DNA damage, thereby allowing for cell cycle arrest via p21Waf1/Cip1/Sdi1 induction, adequate DNA repair, and ultimately enhanced resistance to apoptosis. ^

Chloroflexus spp. and Sulfurihydrogenibium spp. in microbial mats of the Nakabusa hot spring, Japan

Microbial mats develop in a wide range of aquatic habitats, such as geothermal hot springs, hypersaline ponds, marine cold seeps or hydrothermal vents. The Nakabusa hot spring is located in the Nagano Prefecture, Japan (36.3875N, 137.75E), dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65°C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Microbial mat samples were kept in sterile plastic tubes (for molecular analysis) or glass bottles completely filled with hot spring water to avoid oxidation. Samples were transferred to the laboratory on ice and used for physiological experiments within 8h. Quantification of cell biovolumes was carried out based on images of mat sections hybridized with Sulfurihydrogenibium- and Chloroflexi-specific probes, and stained with DAPI. In situ hybridizations (CARD-FISH) of thin matsections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume).

Estudios de especiación de arsénico y acumulación de metales en muestras de interés medioambiental : Arsenic speciation and metal accumulation studies in environmental samples

Se ha estudiado la determinación de especies de arsénico y de contenidos totales de arsénico y metales pesados, específicamente cadmio, cromo, cobre, níquel, plomo y cinc, en muestras de interés medioambiental por su elevada capacidad acumuladora de metales, concretamente algas marinas comestibles y plantas terrestres procedentes de suelos contaminados por la actividad minera. La determinación de contenidos totales se ha llevado a cabo mediante espectrometría de emisión atómica con plasma de acoplamiento inductivo (ICP‐AES), así como por espectrometría de fluorescencia atómica con generación de hidruros (HG‐AFS), para bajos contenidos de arsénico. Las muestras fueron mineralizadas en medio ácido y calentamiento en horno de microondas. Los métodos fueron validados a través de su aplicación a materiales de referencia de matriz similar a la de las muestras, certificados en contenidos totales de los elementos seleccionados. Los resultados obtenidos mostraron su elevada capacidad de bioabsorción, especialmente en relación a los elevados contenidos de arsénico encontrados en algunas especies de algas pardas (Phaeophytas). En las plantas, se calcularon los factores de translocación, acumulación y biodisponibilidad de los elementos estudiados, permitiendo identificar a la especie Corrigiola telephiifolia como posible acumuladora de plomo e hiperacumuladora de arsénico. La determinación de especies de arsénico hidrosolubles en las muestras objeto de estudio, se llevó a cabo por cromatografía líquida de alta eficacia (HPLC) acoplado a ICP‐AES, HG‐ICP‐AES y HG‐AFS, incluyendo una etapa previa de foto‐oxidación. Los métodos desarrollados, mediante intercambio aniónico y catiónico, permitieron la diferenciación de hasta once especies de arsénico. Para el análisis de las muestras, fue necesaria la optimización de métodos de extracción, seleccionándose la extracción asistida por microondas (MAE) con agua desionizada. Asimismo, se realizaron estudios de estabilidad de arsénico total y de las especies hidrosolubles presentes en las algas, tanto sobre la muestra sólida como en sus extractos acuosos, evaluando las condiciones de almacenamiento adecuadas. En el caso de las plantas, la aplicación del diseño factorial de experimentos permitió optimizar el método de extracción y diferenciar entre las especies de arsénico presentes en forma de iones sencillos de mayor movilidad y el arsénico más fuertemente enlazado a componentes estructurales. Los resultados obtenidos permitieron identificar la presencia de arseniato (As(V)) y arsenito (As(III)) en las plantas, así como de ácido monometilarsónico (MMA) y óxido de trimetilarsina (TMAO) en algunas especies. En la mayoría de las algas se encontraron especies tóxicas, tanto mayoritarias (arseniato) como minoritarias (ácido dimetilarsínico (DMA)), así como hasta cuatro arsenoazúcares. Los resultados obtenidos y su estudio a través de la legislación vigente, mostraron la necesidad de desarrollar una reglamentación específica para el control de este tipo de alimentos. La determinación de especies de arsénico liposolubles en las muestras de algas se llevó a cabo mediante HPLC, en modo fase inversa, acoplado a espectrometría de masas con plasma de acoplamiento inductivo (ICP‐MS) y con ionización por electrospray (ESI‐MS), permitiendo la elucidación estructural de estos compuestos a través de la determinación de sus masas moleculares. Para ello, fue necesaria la puesta a punto de métodos extracción y purificación de los extractos. La metodología desarrollada permitió identificar hasta catorce especies de arsénico liposolubles en las algas, tres de ellas correspondientes a hidrocarburos que contienen arsénico, y once a arsenofosfolípidos, además de dos especies desconocidas. Las masas moleculares de las especies identificadas fueron confirmadas mediante cromatografía de gases acoplada a espectrometría de masas (GC‐MS) y espectrometría de masas de alta resolución (HR‐MS). ABSTRACT The determination of arsenic species and total arsenic and heavy metal contents (cadmium, chromium, cooper, nickel, lead and zinc) in environmental samples, with high metal accumulator capacity, has been studied. The samples studied were edible marine algae and terrestrial plants from soils polluted by mining activities. The determination of total element contents was performed by inductively coupled plasma atomic emission spectrometry (ICP‐AES), as well as by hydride generation atomic fluorescence spectrometry (HG‐AFS) for low arsenic contents. The samples studied were digested in an acidic medium by heating in a microwave oven. The digestion methods were validated against reference materials, with matrix similar to sample matrix and certified in total contents of the elements studied. The results showed the high biosorption capacity of the samples studied, especially regarding the high arsenic contents in some species of brown algae (Phaeophyta division). In terrestrial plants, the translocation, accumulation and bioavailability factors of the elements studied were calculated. Thus, the plant species Corrigiola telephiifolia was identified as possible lead accumulator and arsenic hyperaccumulator. The determination of water‐soluble arsenic species in the samples studied was carried out by high performance liquid chromatography (HPLC) coupled to ICP‐AES, HG‐ICP‐AES and HG‐AFS, including a prior photo‐oxidation step. The chromatographic methods developed, by anion and cation exchange, allowed us to differentiate up to eleven arsenic species. The sample analysis required the optimization of extraction methods, choosing the microwave assisted extraction (MAE) with deionized water. On the other hand, the stability of total arsenic and water‐soluble arsenic species in algae, both in the solid samples and in the water extracts, was studied, assessing the suitable storage conditions. In the case of plant samples, the application of a multivariate experimental design allowed us to optimize the extraction method and differentiate between the arsenic species present as simple ions of higher mobility and the arsenic more strongly bound to structural components. The presence of arsenite (As(III)) and arsenate (As(V)) was identified in plant samples, as well as monomethylarsonic acid (MMA) and trimethylarsine oxide (TMAO) in some cases. Regarding algae, toxic arsenic species were found in most of them, both As(V) and dimethylarsinic acid (DMA), as well as up to four arsenosugars. These results were discussed according to the current legislation, showing the need to develop specific regulations to control this kind of food products. The determination of lipid‐soluble arsenic species in alga samples was performed by reversed‐phase HPLC coupled to inductively coupled plasma and electrospray mass spectrometry (ICP‐MS and ESI‐MS), in order to establish the structure of these compounds by determining the corresponding molecular masses. For this purpose, it was necessary to develop an extraction method, as well as a clean‐up method of the extracts. The method developed permitted the identification of fourteen lipid‐soluble arsenic compounds in algae, corresponding to three arsenic‐hydrocarbons and eleven arsenosugarphospholipids, as well as two unknown compounds. Accurate mass measurements of the identified compounds were performed by gas chromatography coupled to mass spectrometry (GC‐MS) and high resolution mass spectrometry (HR‐MS).

Determination of selected pharmaceutical compounds in biosolids bysupported liquid extraction and gas chromatography?tandem massspectrometry

In this work, an analytical method was developed for the determination of pharmaceutical drugs inbiosolids. Samples were extracted with an acidic mixture of water and acetone (1:2, v/v) and supportedliquid extraction was used for the clean-up of extracts, eluting with ethyl acetate:methanol (90:10, v/v).The compounds were determined by gas chromatography?tandem mass spectrometry using matrix-match calibration after silylation to form their t-butyldimethylsilyl derivatives. This method presentsvarious advantages, such as a fairly simple operation for the analysis of complex matrices, the use ofinexpensive glassware and low solvent volumes. Satisfactory mean recoveries were obtained with thedeveloped method ranging from 70 to 120% with relative standard deviations (RSDs) ? 13%, and limitsof detection between 0.5 and 3.6 ng g?1. The method was then successfully applied to biosolids samplescollected in Madrid and Catalonia (Spain). Eleven of the sixteen target compounds were detected in thestudied samples, at levels up to 1.1 ?g g?1(salicylic acid). Ibuprofen, caffeine, paracetamol and fenofibratewere detected in all of the samples analyzed.

The p42 variant of ETS1 protein rescues defective Fas-induced apoptosis in colon carcinoma cells

ETS1 is a cellular homologue of the product of the viral ets oncogene of the E26 virus, and it functions as a tissue-specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. ETS1 controls the expression of critical genes involved in these processes by binding to ets binding sites present in the transcriptional regulatory regions. The ETS1 gene generates two proteins, p51 and a spliced variant, p42, lacking exon VII. In this paper we show that p42-ETS1 expression bypasses the damaged Fas-induced apoptotic pathway in DLD1 colon carcinoma cells by up-regulating interleukin 1β-converting enzyme (ICE)/caspase-1 and causes these cancer cells to become susceptible to the effects of the normal apoptosis activation system. ICE/caspase-1 is a redundant system in many cells and tissues, and here we demonstrate that it is important in activating apoptosis in cells where the normal apoptosis pathway is blocked. Blocking ICE/caspase-1 activity by using specific inhibitors of this protease prevents the p42-ETS1-induced apoptosis from occurring, indicating that the induced ICE/caspase-1 enzyme is responsible for killing the cancer cells. p42-ETS1 activates a critical alternative apoptosis pathway in cancer cells that are resistant to normal immune attack, and thus it may be useful as an anticancer therapeutic.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.

MCD4 Encodes a Conserved Endoplasmic Reticulum Membrane Protein Essential for Glycosylphosphatidylinositol Anchor Synthesis in Yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p’s lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4–174) harbors a single amino acid change in motif 2. The mcd4–174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4–174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4–174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.

The Development of Cell Processes Induced by tau Protein Requires Phosphorylation of Serine 262 and 356 in the Repeat Domain and Is Inhibited by Phosphorylation in the Proline-rich Domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer’s disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.