Disruption of Aldehyde Reductase Increases Group Size in Dictyostelium
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m310539200
ISSN1083-351X
AutoresKaren Ehrenman, Yang Gong, W G Hong, Tong Gao, Wonhee Jang, Debra A. Brock, R. Diane Hatton, James D. Shoemaker, Richard H. Gomer,
Tópico(s)Cellular Mechanics and Interactions
ResumoDeveloping Dictyostelium cells form structures containing ∼20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA– cells grow normally and have decreased glucose levels compared with parental cells. The alrA– cells form long unbroken streams and huge groups. Expression of AlrA in alrA– cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA– cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA– cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA– cells form large groups. alrA– cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA– cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size. Developing Dictyostelium cells form structures containing ∼20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA– cells grow normally and have decreased glucose levels compared with parental cells. The alrA– cells form long unbroken streams and huge groups. Expression of AlrA in alrA– cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA– cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA– cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA– cells form large groups. alrA– cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA– cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size. A fascinating but poorly understood area of biology is how cells create tissues of a specific size (1.Haldane J.B.S. Possible Worlds and Other Papers. Harper & Brothers, New York1928: 20-28Google Scholar, 2.Day S. Lawrence P. Development. 2000; 127: 2977-2987Crossref PubMed Google Scholar, 3.Potter C. Xu T. Curr. Opin. Genet. Dev. 2001; 11: 279-286Crossref PubMed Scopus (123) Google Scholar, 4.Gomer R.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 48-54Crossref PubMed Scopus (58) Google Scholar). A simple model system for this phenomenon is the formation of fruiting bodies in the eukaryote Dictyostelium discoideum, where developing cells form groups of ∼20,000 cells (see Refs. 5.Loomis W.F. Dictyostelium discoideum: A Developmental System. Academic Press, New York1975Google Scholar, 6.Loomis W.F. Curr. Top. Dev. Biol. 1993; 28: 1-46Crossref PubMed Scopus (64) Google Scholar, 7.Devreotes P. Science. 1989; 245: 1054-1058Crossref PubMed Scopus (235) Google Scholar, 8.Schaap P. Dworkin M. Microbial Cell-Cell Interactions. American Society for Microbiology Press, Washington, D. C.1991: 147-178Google Scholar, 9.Firtel R.A. Genes Dev. 1995; 9: 1427-1444Crossref PubMed Scopus (146) Google Scholar, 10.Kessin R.H. Bard J.B.L. Barlow P.W. Kirk D.L. Dictyostelium Evolution, Cell Biology, and the Development of Multicellularity. Cambridge University Press, New York, NY2001Crossref Google Scholar for a review). Dictyostelium normally lives as individual cells that eat bacteria on soil surfaces. As the cells overgrow the bacteria, they starve. The cells then differentiate into either stalk or spore cells and cooperatively form fruiting bodies consisting of a thin, 1–2-mm-high stalk supporting a mass of spores, with the goal of allowing spores to be dispersed by the wind to new patches of soil and a new source of bacteria. For optimal spore dispersal, the fruiting body needs to be as large as possible, with an upper limit dictated by the ability of the stalk to support the spore mass without collapsing or the spore mass sliding down the stalk. Dictyostelium thus have evolved a mechanism to maintain an upper limit to the size of the group of cells that will form an individual fruiting body. Much of the development of Dictyostelium appears to be regulated by secreted factors. When a cell begins starving, it signals to other cells that it is starving by secreting a glycoprotein called conditioned medium factor (11.Mehdy M.C. Firtel R.A. Mol. Cell. Biol. 1985; 5: 705-713Crossref PubMed Scopus (143) Google Scholar, 12.Gomer R.H. Yuen I.S. Firtel R.A. Development. 1991; 112: 269-278PubMed Google Scholar, 13.Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (102) Google Scholar). As the relative density of starving cells increases, the conditioned medium factor concentration concomitantly increases (14.Yuen I.S. Gomer R.H. J. Theor. Biol. 1994; 167: 273-282Crossref PubMed Scopus (36) Google Scholar). When the conditioned medium factor concentration passes a threshold concentration, indicating to the population that there is a high density of starving cells, the cells begin aggregating using relayed pulses of extracellular cAMP as a chemoattractant (15.Jain R. Gomer R.H. J. Biol. 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To elucidate how cells sense the presence of too many cells in a stream and how the subsequent morphogenetic reformation occurs, we isolated a shotgun antisense transformant called smlAas that formed very small fruiting bodies due to excessive stream breakup (20.Spann T.P. Brock D.A. Lindsey D.F. Wood S.A. Gomer R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5003-5007Crossref PubMed Scopus (37) Google Scholar). This transformant, as well as smlA– cells where the corresponding gene was disrupted by homologous recombination, appeared to be oversecreting a factor that when added to starving wild-type cells caused them to form small groups (21.Brock D.A. Buczynski F. Spann T.P. Wood S.A. Cardelli J. Gomer R.H. Development. 1996; 122: 2569-2578Crossref PubMed Google Scholar). Using this as a bioassay, we partially purified the factor and found that it was a 450-kDa complex of polypeptides we named counting factor (CF) 1The abbreviations used are: CF, counting factor; contig, group of overlapping clones; CM, conditioned starvation medium; MES, 2-[N-morpholino]ethanesulfonic acid. (21.Brock D.A. Buczynski F. Spann T.P. Wood S.A. Cardelli J. Gomer R.H. Development. 1996; 122: 2569-2578Crossref PubMed Google Scholar). Disruption of countin, a gene encoding one of the subunits of CF, resulted in cells that formed large fruiting bodies due to streams staying intact and coalescing into large groups (22.Brock D.A. Gomer R.H. Genes Dev. 1999; 13: 1960-1969Crossref PubMed Scopus (108) Google Scholar). Diffusion calculations based on the observed accumulation rate of CF indicated that in general the concentration of a secreted factor such as CF could be used to indicate to cells the number of cells in a stream or group (22.Brock D.A. Gomer R.H. Genes Dev. 1999; 13: 1960-1969Crossref PubMed Scopus (108) Google Scholar, 23.Clarke M. Gomer R.H. Experientia. 1995; 51: 1124-1134Crossref PubMed Scopus (84) Google Scholar). Computer simulations of a stream of cells indicated that decreasing cell-cell adhesion and/or increasing random cell motility would cause a stream to dissipate and subsequently fragment (24.Roisin-Bouffay C. Jang W. Gomer R.H. Mol. Cell. 2000; 6: 953-959Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). If the adhesion then increased or the random motility decreased, the simulations predicted that the dissipated cells would coalesce into a series of groups rather than a single stream. The simulations also predicted that if the adhesion and/or motility were regulated by a secreted factor such as CF, the resulting feedback would allow a very precise control of group size. We found that CF decreases cell-cell adhesion and increases cell motility (24.Roisin-Bouffay C. Jang W. Gomer R.H. Mol. Cell. 2000; 6: 953-959Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25.Tang L. Gao T. McCollum C. Jang W. Vickers M.G. Ammann R. Gomer R.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1371-1376Crossref PubMed Scopus (43) Google Scholar). Decreasing adhesion causes the formation of smaller groups, whereas increasing adhesion or decreasing motility causes the formation of larger groups (24.Roisin-Bouffay C. Jang W. Gomer R.H. Mol. Cell. 2000; 6: 953-959Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25.Tang L. Gao T. McCollum C. Jang W. Vickers M.G. Ammann R. Gomer R.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1371-1376Crossref PubMed Scopus (43) Google Scholar, 26.Kamboj R.K. Lam T.Y. Siu C.H. Cell Regul. 1990; 1: 715-729Crossref PubMed Scopus (31) Google Scholar, 27.Siu C.H. Kamboj R.K. Dev. Genet. 1990; 11: 377-387Crossref PubMed Scopus (37) Google Scholar). Together, the observations suggested that, as predicted by the computer simulations, CF regulates group size by regulating adhesion and motility so as to cause a large stream or group to dissipate. The expression of adhesion molecules and cell motility are regulated by the cAMP pulses that mediate chemotaxis (28.Eckert B. Warren R. Rubin R. J. Cell Biol. 1977; 72: 339-350Crossref PubMed Scopus (37) Google Scholar, 29.Varnum B. Edwards K.B. Soll D.R. J. Cell Biol. 1985; 101: 1-5Crossref PubMed Scopus (54) Google Scholar, 30.Mann S.K.O. Pinko C. Firtel R.A. Dev. Genet. 1988; 9: 337-350Crossref PubMed Scopus (9) Google Scholar, 31.Siu C.H. Lam T.Y. Wong L.M. Biochim. Biophys. Acta. 1988; 968: 283-290Crossref PubMed Scopus (29) Google Scholar, 32.Newell P.C. Biosci. Rep. 1995; 15: 445-462Crossref PubMed Scopus (19) Google Scholar). CF potentiates the cAMP-stimulated cAMP pulse and represses a cAMP-induced cGMP pulse (33.Tang L. Ammann R. Gao T. Gomer R.H. J. Biol. Chem. 2001; 276: 27663-27669Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The signal transduction pathway that allows CF to regulate cAMP signaling, adhesion, and motility is unknown. High levels of glucose cause cells to form large fruiting bodies (34.Garrod D.R. Ashworth J.M. J. Embryol. Exp. Morphol. 1972; 28: 463-479PubMed Google Scholar). CF appears to decrease intracellular glucose levels (35.Jang W. Chiem B. Gomer R.H. J. Biol. Chem. 2002; 277: 31972-31979Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Increasing glucose levels by adding exogenous glucose negates the effect of CF on group size and mimics the effect of decreasing CF on the cAMP-stimulated cAMP pulse, adhesion, and motility. This suggested that CF might regulate glucose levels to regulate group size. Two-dimensional gels of aggregating cells showed a prominent spot that seemed to be most intense in countin– cells and least intense in smlA– cells (25.Tang L. Gao T. McCollum C. Jang W. Vickers M.G. Ammann R. Gomer R.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1371-1376Crossref PubMed Scopus (43) Google Scholar). Two amino acid sequences obtained from tryptic peptides were used to identify sequence fragments from the Dictyostelium sequencing project. When these fragments were assembled, an open reading frame was identified that coded for a predicted protein with strong similarity to aldehyde reductase. The closely related protein aldose reductase converts glucose and NADPH to sorbitol and NADP (36.Lee H. Yeast. 1998; 14: 977-984Crossref PubMed Scopus (47) Google Scholar). Both aldose reductase and aldehyde reductase reduce a wide variety of aldehydes, and their exact functions within cells are still unknown. Aldose reductase is thought to be responsible for some of the complications of diabetes such as neuropathy, cataracts, and retinopathy (37.Dunlop M. Kidney Int. Suppl. 2000; 77: 3-12Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 38.Cooper M. Diabetologia. 2001; 44: 1957-1972Crossref PubMed Scopus (327) Google Scholar). The high levels of glucose in diabetics cause the production of high levels of sorbitol. Sorbitol acts as an osmolyte, and the high levels of sorbitol are then thought to cause a high osmotic pressure within lens and retinal cells, which then causes cellular damage. Because glucose seems to play a role in a cell number-counting signal transduction pathway and because aldose reductase and aldehyde reductase may affect glucose levels, we have examined the function of aldehyde reductase in Dictyostelium cells. Sequence Assembly—Preliminary sequence data were obtained from the Dictyostelium BLAST site (available on the World Wide Web at dicty.sdsc.edu/) using the raw reads and contigs provided by the Baylor Sequencing Center and the Institute of Biochemistry (Cologne, Germany) together with the Institute of Molecular Biotechnology (Jena, Germany) and the EUDICT consortium. Sequences were assembled and analyzed using software from the Genetics Computer Group (Madison, WI). Genomic DNA Extraction—2 × 107 vegetative cells were collected by centrifugation and resuspended in 0.5 ml of GL buffer (120 mm NaCl, 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 0.5% SDS). 5 μl of an RNase mixture (50 units/ml RNase A and 100 units/ml RNase T1) (Roche Applied Science) was added to the cells and incubated for 20 min at 55 °C. 50 μl of 10 mg/ml proteinase K (Roche Applied Science) was added, and the mixture was incubated for an additional 2 h at 55 °C. An equal volume of 1 m Tris (pH 8.0)-buffered phenol was added, and the mixture was gently vortexed. After centrifugation at 10,000 × g for 5 min, the aqueous phase was similarly treated with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). The aqueous phase was mixed with 0.1 volumes of 3 m sodium acetate (pH 5.2) and 1 volume of isopropyl alcohol. After centrifugation at 13,000 × g for 15 min, the pellet was washed with 70% ethanol, dried, and resuspended in 50 μl of TE (10 mm Tris-HCl, pH 8.0, and 1 mm EDTA, pH 8.0). Disruption of alrA by Homologous Recombination—PCR was done using Ax4 genomic DNA as a template to create DNA fragments flanking the aldehyde reductase gene. The left arm primers were TCGCGCCGCGGCTCAACTTCACCAGTTGTCATTTTACC, which added a SacII site to the sequence, and TATCGGCCGAAAACGATGTTCTTGTGTGTTTGG, which added an EagI site. This generated a 1.0-kb band. The right arm primers were ATCGCGTCGACGTGAAGATAATGGTGCACAC, adding a HincII site, and ATAGGGCCCGAGCTGGTCTAAGGGG, adding an ApaI restriction site. This generated a 1.2-kb fragment. The backbone of the construct used to generate the aldehyde reductase knockout was pBB, pBluescript SK(+) (Stratagene, La Jolla, CA) containing a 1.4-kb blasticidin resistance cassette inserted into the XbaI-HindIII sites (39.Sutoh K. Plasmid. 1993; 30: 150-154Crossref PubMed Scopus (169) Google Scholar, 40.Adachi H. Hasebe T. Yoshinaga K. Ohta T. Sutoh K. Biochem. Biophys. Res. Commun. 1994; 205 (Correction (1995) Biochem. Biophys. Res. Commun. 208, 1181): 1808-1814Crossref PubMed Scopus (155) Google Scholar). The left arm fragment generated by the above PCR was digested with SacII and EagI, and the right arm fragment was digested with HincII and ApaI. These fragments were then ligated into the corresponding sites of pBB to generate pARKO. The SacII-ApaI fragment of pARKO was then used to transform Ax4 cells, and blasticidin-resistant cells were selected following Kuspa and Loomis (41.Kuspa A. Loomis W.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8803-8807Crossref PubMed Scopus (398) Google Scholar). The resulting cell strain was designated ARKO. Expression of AlrA in alrA– Cells—To express AlrA in Dictyostelium cells, the expression vector pDXA3-C (42.Manstein D. Schuster H. Morandini P. Hunt D. Gene (Amst.). 1995; 162: 129-134Crossref PubMed Scopus (192) Google Scholar) was altered by Dr. William Deery to remove the ATG upstream of the cDNA insertion site. The sequence of the altered vector from the HindIII site (underlined) to the KpnI (italicized) site is 5′-AAGCTTAAAGTTCGAATTCAAAGGTACC-3′, and this vector was designated pDXA3-D. A SMART RACE cDNA amplification kit (Clontech Laboratories, Palo Alto, CA) was used to generate cDNA from Ax4 RNA. This cDNA was then used to obtain alrA cDNA using the forward primer 5′-GGGGTACCATGGAACCATCATTTAAATTATC-3′ (containing a KpnI restriction site and the first 23 nucleotides of the coding region of alrA) and the reverse primer 5′-CCCTCGAGTTAATTGAAAAGTGGTACACCC-3′ (containing an XhoI restriction site and the last 19 nucleotides of the coding region and the termination codon of alrA). This cDNA fragment was digested with KpnI and XhoI and ligated into the similarly digested vector. The cDNA in the resultant vector pAlrAOE was then sequenced to verify that there were no errors. alrA– cells were then transformed with pAlrAOE and the helper plasmid pREP following Manstein et al. (42.Manstein D. Schuster H. Morandini P. Hunt D. Gene (Amst.). 1995; 162: 129-134Crossref PubMed Scopus (192) Google Scholar), and transformants were selected with 2.5 μg/ml G418 and then checked to verify blasticidin resistance. The resulting selected strain was designated ARKOA2. Expression of AlrA in the ARKOA2 cells was verified by staining Western blots of whole cell lysates with anti-AlrA antibodies (see below). Cell Culture, Group Number Assays, and Western Blots—D. discoideum Ax2 and Ax4 wild types, smlA– (strain HDB7YA) cells, and cells with a disruption of the ctnA gene (strain HDB2B/4) (referred to in this and previous work as countin– cells) were grown as previously described (21.Brock D.A. Buczynski F. Spann T.P. Wood S.A. Cardelli J. Gomer R.H. Development. 1996; 122: 2569-2578Crossref PubMed Google Scholar, 22.Brock D.A. Gomer R.H. Genes Dev. 1999; 13: 1960-1969Crossref PubMed Scopus (108) Google Scholar). Photography of fruiting bodies followed Brock et al. (43.Brock D.A. Hatton R.D. Giurgiutiu D.-V. Scott B. Ammann R. Gomer R.H. Development. 2002; 129: 3657-3668Crossref PubMed Google Scholar). Streams and aggregates on SM/5 agar plates with Klebsiella aerogenes bacteria were photographed with a Nikon D1 camera with a macro lens. Cells were developed on filter pads as described in Jain et al. (13.Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (102) Google Scholar). Preparation of conditioned starvation medium (CM) and staining of Western blots with anti-CF50 and anti-countin antibodies followed Brock et al. (43.Brock D.A. Hatton R.D. Giurgiutiu D.-V. Scott B. Ammann R. Gomer R.H. Development. 2002; 129: 3657-3668Crossref PubMed Google Scholar). Cell-mixing (synergy) experiments were performed following Brock et al. (21.Brock D.A. Buczynski F. Spann T.P. Wood S.A. Cardelli J. Gomer R.H. Development. 1996; 122: 2569-2578Crossref PubMed Google Scholar). Recombinant countin was prepared following Gao et al. (44.Gao T. Ehrenman K. Tang L. Leippe M. Brock D.A. Gomer R.H. J. Biol. Chem. 2002; 277: 32596-32605Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and recombinant CF50 was prepared following Brock et al. (43.Brock D.A. Hatton R.D. Giurgiutiu D.-V. Scott B. Ammann R. Gomer R.H. Development. 2002; 129: 3657-3668Crossref PubMed Google Scholar). The effect of recombinant proteins on group size was assayed in submerged culture following Brock and Gomer (22.Brock D.A. Gomer R.H. Genes Dev. 1999; 13: 1960-1969Crossref PubMed Scopus (108) Google Scholar). Reverse Transcriptase-PCR and Northern Blots—RNA was extracted from 2 × 107 cells growing in HL5 at a concentration of 2–4 × 106 cells/ml using the RNeasy total RNA isolation kit (Qiagen, San Clarita, CA). cDNA was synthesized using the Prostar Ultra HF reverse transcriptase-PCR system (Stratagene, La Jolla, CA) utilizing Maloney murine leukemia virus reverse transcriptase. PCR was done using the cDNA as a template. The primers GCTGTTGAAGTTGCTCTCGATGCTG and GTACACCCCAGAATTTAGCTGGATC were used to amplify a 780-bp aldehyde reductase sequence. As a control, primers GATGGATCACAATAGATATTCAGCAG and TCCGACTGAATGGGGTTTGCTATCAT were used to amplify a 217-bp sequence from sslA (accession number AAM34288). RNA isolation from vegetative cells and Northern blots was performed following Brock et al. (43.Brock D.A. Hatton R.D. Giurgiutiu D.-V. Scott B. Ammann R. Gomer R.H. Development. 2002; 129: 3657-3668Crossref PubMed Google Scholar). The 780-bp alrA fragment described above was used as a probe. Anti-aldehyde Reductase Western Blots and Cell Fractionation—A total of 1 × 106 cells were collected by centrifugation and resuspended to 50 μl in Laemmli sample buffer. Following the ECL protocol (Amersham Biosciences), the blot was then stained with a 1:2,000 dilution of a 1.3 mg/ml solution of affinity-purified antibody made against the aldehyde reductase-specific peptide CWNTFHKKEHVRPALER (Bethyl Laboratories Inc., Montgomery, TX). For cell fractionation, Ax4 cells were collected by centrifugation and either used immediately or starved in shaking culture in PBM (20 mm KH2PO4/K2HPO4, 1 mm MgCl2, 0.01 mm CaCl2, pH 6.1) for 3 h as described above. 1 × 109 cells were collected and resuspended to 1 × 108 cells/ml in ice-cold MESES buffer (20 mm MES, pH 6.5, 1 mm EDTA, and 0.25 m sucrose). Cells were lysed through a 5.0-μm cameo 25N-syringe filter (Osmonics/MSI, Westborough, MA). Cell fractionation was done using centrifugation following Brock et al. (21.Brock D.A. Buczynski F. Spann T.P. Wood S.A. Cardelli J. Gomer R.H. Development. 1996; 122: 2569-2578Crossref PubMed Google Scholar). Aldehyde Reductase Assay—A 5.0-μm syringe filter was washed with 5 ml of PBM, and then 3 ml of cells at 5 × 107 cells/ml were lysed by a single passage of these cells through the filter. For some assays, the lysate was clarified by centrifugation at 19,000 × g for 1 min. The aldehyde reductase activity was determined by the decrease of NADPH absorption at 340 nm (relative to the background absorption at 400 nm) at room temperature using dl-glyceraldehyde, water, or d-glucose as substrates for the enzyme (45.Gabbay K. JH K. Methods Enzymol. 1975; 41: 159-165Crossref PubMed Scopus (12) Google Scholar). 500 μl of 0.135 m Na2HPO4/KH2PO4, pH 6.2, 200 μl of water, 100 μl of 0.73 mm NADPH tetrasodium salt (Sigma), and 100 μl of 50 mm mercaptoethanol were added to 100 μl of cell lysate. This was placed in a cuvette, and then either 25 μl of 0.04 m dl-glyceraldehyde, 4 m d-glucose, or water was added. The enzymatic activity over the course of 2 min was determined for each substrate by Z = (AT = 0340 – AT = 0400) – (AT = 2340 – AT = 2400). Following Gabbay and Kinoshita (45.Gabbay K. JH K. Methods Enzymol. 1975; 41: 159-165Crossref PubMed Scopus (12) Google Scholar), 1 unit of activity was defined as the ability to oxidize 1 μmol of NADPH/hour. Given an extinction coefficient for NADPH of 6.22 OD/cm/mm, the units of activity were 4,823 × Z. The values for water were then subtracted from those of glyceraldehyde and glucose. Protein concentrations were determined with a Bio-Rad protein assay in comparison with a bovine serum albumin standard dilution curve. Adhesion and Motility Assays—Ax4 and alrA– cells were grown and assayed for adhesion following the protocol of Desbarats et al. (46.Desbarats L. Brar S.K. Siu C.H. J. Cell Sci. 1994; 107: 1705-1712PubMed Google Scholar) as modified by Roisin-Bouffay et al. (24.Roisin-Bouffay C. Jang W. Gomer R.H. Mol. Cell. 2000; 6: 953-959Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), allowing the disaggregated cells 2 min to aggregate before scoring for single cells. To measure motility, midlog phase cells growing in HL5 were collected by centrifugation, resuspended and washed in PBM, and resuspended to 1 × 107 cells/ml. 200 μl of cells were starved on a filter pad. At 6, 8, 10, and 12 h, cells were harvested and diluted to 2 × 105 cells/ml in PBM, and 200 μl of cells was placed in a well of an 8-well slide. For 0-h motilities, midlog cells in HL5 were diluted to 2 × 105 cells/ml with HL5, and 200 μl of cells was placed in a well of an 8-well slide. For all of the time points, cells were allowed to settle for 15 min, and motility was then measured by videotaping the cells following Yuen et al. (16.Yuen I.S. Jain R. Bishop J.D. Lindsey D.F. Deery W.J. Van Haastert P.J.M. Gomer R.H. J. Cell Biol. 1995; 129: 1251-1262Crossref PubMed Scopus (66) Google Scholar). The approximate distance moved by a cell was measured in 1-min increments over 10 min. Computer Simulations—The JAVA computer simulations used the program described in Roisin-Bouffay et al. (24.Roisin-Bouffay C. Jang W. Gomer R.H. Mol. Cell. 2000; 6: 953-959Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) with the following modifications. The aggregation stream at the beginning of the simulation was 2000 cells in length and 17–22 cells in width. For the distribution of cell motilities, we used the actual distribution of motilities observed with either wild-type or alrA– cells. The cell-cell adhesions were set as the cell-cell adhesions observed with wild-type or alrA– cells. Glucose and Osmolality Assays—Glucose levels were measured as described by Jang et al. (35.Jang W. Chiem B. Gomer R.H. J. Biol. Chem. 2002; 277: 31972-31979Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). To measure osmolality, ∼2 × 108 vegetative cells or cells starved at 5 × 106 cells/ml for 6 h in PBM in shaking culture were collected by centrifugation. The pellets were briefly recentrifuged, the remaining supernatant was removed, and the pellets were frozen at –80 °C. The pellets were thawed, and the osmolality of 50 μl of a 1:1 mixture of the lysed cells and distilled water was measured with a model 5004 Micro Osmometer (Precision Systems, Natick, MA). A standard curve was constructed using distilled water and dilutions of a 100-mosmol/kg H2O standard solution (Precision Systems), and the osmolality of the original cell lysate was calculated. Protein concentrations were measured using a Bio-Rad protein assay and bovine serum albumin for a calibration curve. Metabolite Analysis—For vegetative cell samples, cells were grown to 1–2 × 106 cells/ml in HL5, and for development cells were starved at 5 × 106 cells/ml in PBM in shaking culture. A total of 1 × 109 cells were collected by centrifugation at 1,500 × g for 5 min. The pellets were resuspended in ∼5 ml of the remaining supernatant, and this was recentrifuged. The supernatants were carefully aspirated, and the pellets were frozen at –80 °C. After 1–4 days, the pellets were thawed, and 2 ml of water was added. This mixture was vortexed and then clarified by centrifugation at 23,000 × g for 10 min at 4 °C. 1.2 ml of the slightly cloudy supernatant was then frozen on dry ice. After thawing, acetone was added to 50%, and the samples were processed for azetropic dehydration and trimethylsilylation of compounds as described in Shoemaker and Elliot (47.Shoemaker J.D. Elliot W.H. J. Chromatogr. 1991; 562: 125-138Crossref PubMed Scopus (122) Google Scholar) starting at the acetone addition step. Gas chromatography/mass spectrometry of the processed samples was performed as described by Shoemaker
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