tol 3-phosphate (PI(3)P), phosphatidylinositol 3,4- bisphosphate. (PI(3,4)P2) or phosphatidylinositol. 3,4,5-trisphosphate (PIP3). The circumstances and ...
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Journal of Cell Science 111, 283-294 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS4436
Disruption of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphosphate and [32P]phosphatidylinositol trisphosphate levels, alters Factin distribution and impairs pinocytosis Kemin Zhou2, S. Pandol3, G. Bokoch2 and A. E. Traynor-Kaplan1,* 1Department of Medicine, The University of California San Diego, The Whittier Institute, 9894 Genesee Ave., La Jolla, CA 920370983, USA 2Department of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA, USA 3Department of Research and Development, VA Hospital, Los Angeles, USA
*Author for correspondence at Inologic, Inc., 43012 S.E. 108th St, North Bend, WA. 98045, USA
Accepted 28 October 1997: published on WWW 23 December 1997
SUMMARY To understand how phosphatidylinositol 3-kinase (PI3K) modulates cell structure and function, we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik1∆2∆) missing two (DdPIK1 and 2) of three PI3K genes, which are homologues of the mammalian p110 subunit. Levels of [32P]phosphatidylinositol 3,4 bisphosphate (PI(3,4)P2) and [32P]phosphatidylinositol trisphosphate (PIP3) were reduced in pik1∆2∆, which had major defects in morphological and functional correlates of macropinocytosis. This was accompanied by dramatic deficits in a subset of F-actin-enriched structures such as
circular ruffles, actin crowns and pseudopodia. Although pik1∆2∆ were mobile, they failed to aggregate into streams. Therefore we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI(3,4)P2, regulate the organization of actin filaments necessary for circular ruffling during macropinocytosis, the extension of pseudopodia and the aggregation of cells into streams, but not the regulation of cell motility.
INTRODUCTION
subfamily. DdPIK4 encodes a PI-4 kinase. The amino acid sequences of the DdPIK1 and 2 products resemble the p110α and β isoforms encoded by the mammalian genes, whereas the sequence of the DdPIK3 product resembles the mammalian p110γ isoform (Stoyanov et al., 1995). DdPIK5 complements the defects of vps34 gene disruption in yeast and is essential for Dictyostelium to use bacteria as food (Zhou et al., 1995). In mammals, the p110 catalytic subunits α and β are regulated through a p85 subunit. SH2 domains on p85 associate with phosphotyrosine residues on a pleiomorphic array of proteins and facilitate the translocation of the p110/p85 complex from the cytosol to the membrane. In contrast, p110γ appears to be activated through heterotrimeric G proteins (Stoyanov et al., 1995). It is not yet known whether the corresponding Dictyostelium gene products are regulated in an analogous fashion to p110γ. The PI3K pathway has been implicated in the regulation of a multitude of intracellular events including mitogenesis (Carraway et al., 1995), membrane ruffling (Kotani et al., 1994), chemotaxis (Wennstrom et al., 1994), apoptosis (Yao et al., 1990), activation of neutrophils and oxygen radical formation (Ding et al., 1995; Traynor-Kaplan et al., 1989), receptor internalization (Joly et al., 1994; Kapeller et al., 1993), pinocytosis (Kotani et al., 1995) and
Enzymes in the phosphatidylinositol-3 kinase (PI3K) family phosphorylate inositol phospholipids on the D-3 position of the myo-inositol ring, resulting in the production of 3phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 3,4bisphosphate (PI(3,4)P2) or phosphatidylinositol 3,4,5-trisphosphate (PIP3). The circumstances and characteristics of the rise in the levels of these products following receptor activation in a variety of cell types suggest that they are intracellular signals. However the relative roles of the individual lipids and the mechanisms by which they are differentially produced remain obscure. In at least one instance, the vsp34 gene product appears to be a PI3K with a restricted substrate specificity, since PI(3)P is the only demonstrated product either in intact cells or in a cell-free assay. Therefore, the differential production of the various D-3 phosphoinositides may be due to different enzyme isoforms with varying substrate specificity. Four PI3K genes have been identified in Dictyostelium: DdPIK5, which encodes a member of the PI3K-I subfamily, and DdPIK1, 2 and 3, which encode members of the PI3K-II
Key words: Actin cytoskeleton, Pinocytosis, PI-3 kinase knockout mutants, PIP3
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neurite outgrowth (Kimura et al., 1994). Multiple, putative downstream targets of the PI3K-II family have been identified, such as AKT/protein kinase B (Burgering and Coffer, 1995), protein kinase C (Nakanishi et al., 1993; Singh et al., 1993), AKT and the small GTPases, Rac and Ras (Hawkins et al., 1995; Hu et al., 1995; Kotani et al., 1995; Yamauchi et al., 1993). It is difficult to ascertain whether the above phenomena are directly or indirectly linked to PI3K activity and/or specific phospholipid products, although AKT/PKB appears to be downstream of PI(3,4)P2 and not PIP3 (Franke et al., 1997; Klippel et al., 1997). Although many targets may exist for the 3-phosphorylated phosphoinositides, most of the functions associated with PI3K activity require an intact, normally functioning cytoskeletal network. To address these issues, we have generated a Dictyostelium mutant (pik1∆2∆) lacking two of three identified PI3K-II genes. As reported previously (Zhou et al., 1995), the double-knockout cells are smaller and divide more slowly but can form fruiting bodies and produce spores. In a recent study with pik1∆2∆, Buczynski and coworkers presented data which indicated that PI-3 kinase activity assayed by PI(3)P formation was not diminished (Buczynski et al., 1997). However, any defect in pik1∆2∆-mediated PI3P formation would be likely to be masked by the abundance of DdPIK5 in their preparation. Because DdPIK1 and 2 more closely resemble mammalian p110 isoforms than the vps34 gene product, PI(3,4)P2 or PIP3 are more likely to be diminished in the mutant. Here, we demonstrate that PI(3,4)P2 and PIP3 levels are reduced in the pik1∆2∆ strain relative to wild-type and complemented strains. Moreover, pinocytosis and phagocytosis of autoclaved bacteria were incapacitated in double-knockout strains while motility and cyclic AMP (cAMP)-induced cAMP production were not impaired. Furthermore, specific actin cytoskeletal defects were apparent that were consistent with the functional deficits. Therefore, we provide evidence that deletion of specific PI3K genes can result in reduced levels of PIP3 and PI(3,4)P2 in Dictyostelium. Furthermore, this corresponds to changes in cytoskeletal structures containing F-actin, which may explain defects in endocytosis documented here as well as abnormalities in cell size and division previously reported.
MATERIALS AND METHODS Materials The double null mutant, pik1∆2∆, was derived from D. discoideum wild-type strain A×3 as described elsewhere (Zhou et al., 1995). [32P]Orthophosphate was obtained from New Enghand Nuclear. All organic solvents were obtained from Fisher Scientific and were HPLC grade. Molecular biology methods Plasmid pPIK2 was constructed from a genomic DNA clone that contains a 0.8 kb 5′-untranslated region and a 3.7 kb 5′-portion ORF, and a partial DdPIK2 cDNA that codes for the 3′-portion 2.2 kb ORF of DdPIK2 (Zhou et al., 1995). The chimera was fused at the NdeI restriction site. A Tn5-Neo expression cassette (K. Zhou, unpublished) was inserted, as a Klenow-treated XhoI-EcoRV fragment, into the SmaI site in the 5′ polylinker region of pPIK2N. Transcription of Tn5-Neo gene is driven by a vegetative-specific promoter H5b-A (Singleton et al., 1991). For Southern analysis of complemented strains we used a probe from the 5′ SnaBI site to the HindIII site, which covers the majority
of the DdPIK2 coding region. The procedures are described (Zhou et al., 1995). Cell growth, development and transformation Dictyostelium cells were grown in a rich medium HL5 on Petri dishes. Klebsiella aerogenes plates were used for observing growth and development of the different strains. DNA was introduced into Dictyostelium cells by electroporation. Detailed techniques are described elsewhere (Dynes et al., 1994; Hadwiger and Firtel, 1992; Mann and Firtel, 1991). Mutant cultures were used for ten passages or less because later-passage cells began to revert to the wild-type phenotype. Stream assay HL5 medium was removed from a plate with confluent Dictyostelium cells and replaced with 8 ml of 12 mM Na/K-PO4 buffer. This initiates starvation, causing the Dictyostelium to form aggregation streams. Chemotaxis For the agar well assay, the procedures were exactly as described before (Mann and Firtel, 1991). Axenicly grown or 6 hour-starved cells were deposited 3 mm from the edge of the well with a thin, Pasteur pipette. Chemotaxis was initiated by the addition of 1 mM folate or cAMP into the well. Movement of the cells was monitored for 3 hours. For the radial assay (Browning et al., 1995), a cell slurry was deposited with a Pasteur pipette onto the surface of a thin layer of agarose containing 1 mM to 100 mM cAMP or folate. Motility of cells was monitored for 3 hours. Endocytosis Analysis of endocytosis was performed essentially as described (Vogel, 1987). Pinocytosis was assayed as fluorescence of Dictyostelium cells cultured in HL5 medium containing 1.5 mg/ml FITC-labeled dextran. Fluorescence at 490 nm excitation and 520 nm emission was determined with a PERKIN ELMER 650-10S Fluorescence Spectrophotometer. Dictyostelium cell number in endocytic medium was determined with a hemocytometer. Consumption of live bacteria was monitored as a decrease in OD590 in a coculture of Dictyostelium and E. coli B/r in 12 mM Na/K-PO4 with shaking at 150 rpm. Dictyostelium cells used for these studies were washed off plates and cultured for 12-24 hours in shaking culture before mixing with the bacteria. For comparison, Dictyostelium cells from different strains were inoculated to the same initial OD590. Phagocytosis was analyzed as the accumulation of fluorescent bacteria E. coli B/r in Dictyostelium cells in 20 mM phosphate buffer, pH 6.3. following the method of Cohen et al. (1994). Mathematical analysis of pinocytosis data Relative cell fluorescence was converted to the volume of fluid marker uptake by comparison to a standard curve of fluorescence versus volume of medium in the same sample. The amount of liquid uptake at different time points was fitted to equation 1. We tested this equation by fitting it to previously published pinocytosis data (O’Halloran and Anderson, 1992). A Macintosh program that calculates all pinocytosis parameters based on the minimal sum of squares of deviation of observed data is available upon request. Analysis of D-3 phosphorylated phosphoinositides Log-phase confluent Dictyostelium cells were grown in low phosphate medium, MES-HL5, for at least 12 hours before culturing the cells in MES-FM, a synthetic medium without phosphate (Franke and Kessin, 1977) for 12 hours. Afterwards, the cells were either kept in this medium, or starved in 12 mM Na/K-PO4 for 5 hours. Cells were labeled with 0.25 mCi [32P]orthophosphate per plate for 3 hours prior to lipid extraction. The labeling was stopped following aspiration of labeling buffer with the addition of ice-cold methanol/2.4 N HCl (1/1, v/v) to the plate and scraping. Subsequent extraction, thin layer chromatography,
PI3K-II function revealed by gene knockout in Dictyostelium deacylation and HPLC analyses were exactly as described (TraynorKaplan et al., 1989). cAMP assay Dictyostelium cells were grown to confluence on Petri dishes in HL5 medium. The day of the assay the medium was aspirated and replaced with 12 mM Na/K PO4. After 5 hours the cells had begun to organize into concentric rings or spirals, at which point they were washed from the plates, pelleted and resuspended in phosphate buffer to 1×108 cells/ml. They were then put on ice for 1-2 hours for synchronization. The cells were then stimulated with 10 µM deoxy-cAMP and samples were collected at various times from 15 seconds to 5 minutes for quantitation of cAMP levels using a kit (Amersham, TRK432). Light scattering assay Confluent cultures of Dictyostelium as described above were washed and resuspended in phosphate buffer at a concentration of 5×107 cells/ml. Right angle light scattering was monitored in real time using an SLM 8000 spectrofluorimeter (SLM Instruments, Urbana, IL.) as described previously (Sklar et al., 1985). Electron microscopy Cells grown on Permanox Lux plates or in shaking culture were washed once quickly with 12 mM Na/K-PO4 buffer and fixed immediately in modified Karnovsky’s fixative: 2% paraformaldehyde, 1% glutaraldehyde, 5 mM CaCl2, 0.1 M sodium cacodylate buffer, pH 7.2. The fixing process took either 1 hour at room temperature or 12 days at 4°C. The subsequent procedures were exactly as described (O’Konski and Pandol, 1990). F-actin staining Cells on coverslips were fixed with 2% paraformaldehyde in PBS for 10 minutes at room temperature, followed by three 5 minute washes with PBS. The fixed cells were permeabilized with 0.05% Triton X100 in PBS for 1.5 minutes and washed thrice as before. Then cells on each coverslip were stained with 0.2-0.5 units of rhodaminephalloidin in PBS for 20 minutes at room temperature. Finally, the cells were washed three times as before and mounted on a glass slide for fluorescence microscopy.
RESULTS A Dictyostelium double-knockout strain (pik1∆2∆) lacking two PI3K-IIa genes has defects in growth and forms abnormally shallow and diffuse plaques when grown on a bacterial lawn (Zhou et al., 1995). Here we report the molecular and cellular characterization of pik1∆2∆. First, by complementation, we demonstrated that the double-knockout phenotype is not caused by random mutation. Complementation of pik1∆2∆ by the DdPIK2 gene Knockout of each of the three known PI3K-II genes, DdPIK1, DdPIK2 and DdPIK3, had no detectable effects on the Dictyostelium phenotype (Zhou et al., 1995). Therefore, introducing either DdPIK1 or DdPIK2 into pik1∆2∆ would be expected to restore the wild-type-like phenotype if there are no random background mutations in the double-knockout strain. The parental strain, JH10 (Thymidine−), has a single wild-type DdPIK2 locus that gave a 1.7 kb and a 4.0 kb SnaBl-HindIII fragment in Southern analysis. The double-knockout strain has one (isolate 8452) or two copies (isolate 4825) of the disrupted DdPIK2 gene, which gave a 5.2 kb fragment. All four randomly chosen pPIK2-transformants had both the wild-type and the disrupted genes. Based on the intensity of the
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complemented DNA bands, only a single copy of the DdPIK2 gene was incorporated into the genome. Complemented strains grew and developed on a bacterial lawn in a manner that was similar to the wild-type strain, although complemented cells tended to be slightly larger. In contrast, its parental strain, pik1∆2∆, grew slowly as an abnormally shallow and diffuse plaque on bacteria, and fruiting body formation was stunted and severely delayed (Zhou et al., 1995). Furthermore, in all cases described below, complementation either restored or exceeded the wild-type-like phenotypes. Reduced [32P]PI(3,4)P2 and [32P]PIP3 levels in pik1∆2∆ Phospholipid levels were compared in wild type (AX3), mutant (pik1∆2∆), and complemented cells grown in medium or starved for 5-6 hours. Cells were labeled with [32P]orthophosphate as described in Materials and methods and the lipids were extracted and analyzed by thin layer chromatography and HPLC. [32P]PI(3,4)P2 and [32P]PIP3 levels in cells grown in medium were reduced in pik1∆2∆ compared to complemented strains or that of AX3 (Fig. 1). Starvation of Dictyostelium cells causes them to initiate multicellular development. To determine whether PI3K-II plays a role in this process, we examined the levels of phosphoinositides in cells starved for 5-6 hours. In starved cells, levels of [32P]PI(3,4)P2 in pik1∆2∆ were comparable to those of AX3 and 75% of levels in the complemented cells, whereas [32P]PIP3 levels were approximately 30% of the complemented levels and 50% of those in wild type (Fig. 1). Because others have suggested that PI3K may play a role in chemotaxis, we questioned whether exposure of wild-type cells to the chemoattractant cAMP elicited changes in D-3 phosphoinositide levels. However, phosphoinositide levels in starved, wild-type cells were comparable to those in Fig 1 and were not elevated in cells exposed to cAMP for varying lengths of time (data not shown). The double-knockout strain has defects in streaming that can be rescued by complementation with the DdPIK2 gene After 4-5 hours of starvation, Dictyostelium cells begin pulsatile cAMP secretion, creating centers for aggregation. The pulsatile cAMP signal induces chemotaxis in surrounding cells, which respond with more cAMP secretion, creating a cAMP gradient attracting the outermost cells toward the center. Thus, a signal generated by a single cell can be transmitted several centimeters, causing aggregation and formation of aggregation streams that are visible without the aid of a microscope. The double-knockout cells were smaller and the formation of aggregation streams was perturbed. After 5-6 hours of starvation, concentric rings or spirals of cells could be detected on the plates containing both wild type and knockout mutants. These patterns have been reported to correspond to early stages of chemotaxis (Gerisch and Hess, 1974). After 12 hours of starvation, most AX3 and complemented cells formed aggregation streams (Fig. 2C,D), whereas pik1∆2∆ cells had not begun to aggregate (Fig. 2E). After 20 hours of starvation, most AX3 or pPIK2/pik1∆2∆ cells had aggregated into mounds (Fig. 2F). In contrast, pik1∆2∆ cells formed small and numerous aggregates only much later (Fig. 2G). Indeed, the cells had still not aggregated in some areas after 3 days and streams never
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A
B
C Fig. 1. [32P]PI(3,4)P2 and PIP3 levels are reduced in the double-knockout strains. (a,b) Chromatograms of an HPLC separation of deacylated [32P]labeled phospholipids extracted from Dictyostelium. Radioactivity was measured with an on-line radioisotope detector. The abscissa is the retention time in minutes. The ordinate shows the 32P incorporation in counts per minute. Traces were normalized to [32P]PIP2. (a) Chromatogram showing full scale. Based on the retention times of standards, Peak A is identified as glycero ( Gro)-PI4P, peak B as Gro-PI(3,4)P2, peak C as Gro-PI(4,5)P2 and peak D as Gro-PI (3,4,5)P3. The peak between 55 and 60 minutes is Ins(1,4,5)P3. (b) The expanded chromatogram in the region where Gro-PIP3 elutes. (c) Data for the effects of growth conditions on phosphoinositide levels. The open bars represent phospholipids from cells maintained in growth medium whereas the hatched bars represent phospholipid levels from cells in phosphate buffer. AX3, wild type; C, complemented. became apparent. One explanation for this phenomenon is that pik1∆2∆ cells generate a chemotactic signal and are able to move short distances but do not form aggregation streams. Further evidence that they are capable of generating a chemotactic signal comes from the observation that 2-deoxycAMP triggered comparable levels of cAMP production in both mutant and wild-type cells starved for 5 hours. Double-knockout cells are motile but lack mound formation The inability to form aggregation streams could be due to any of the following: (1) inability to detect the cAMP gradient, (2) motility defect, (3) defect in cAMP-stimulated cAMP production, release and accumulation, (4) the absence of a cAMP gradient due to failure to hydrolyze cAMP or (5) the inability to form stable cell-cell associations. In the studies of Buczynski and coworkers (1997), double-knockout cells responded to a micropipette containing cAMP more rapidly than wild type, indicating that the rate of response to chemoattractants and the rate of chemotaxis is faster in the mutants. Therefore, these cells appear to be able to detect a cAMP gradient and have no motility deficit (causes 1 and 2). Defects in causes 3 and 4 would be apparent in assays where the cells themselves
participate in the formation of the gradient (Browning et al., 1995). A slurry of cells was deposited on a thin layer of agarose that contains cAMP or folate (1 mM to 0.1 mM). In the case of folate, vegetative cells were used, whereas in the case of cAMP, cells were starved for 5 hours prior to the assay (See Materials and methods). The double-knockout cells migrated at the same rate as AX3 and pik1∆2∆/pPIK2 on both folate and cAMP, suggesting that the ability of the knockout cells to generate a gradient was not impaired. However, there was a fundamental difference in the manner in which the double-knockout cells migrated. During migration toward cAMP, AX3 and pik1∆2∆/pPIK2 formed slug-like structures, whereas pik1∆2∆ formed a more uniform, outwardly migrating layer (Fig. 3). The same was true for folate (data not shown). However, pik1∆2∆ cells continued migrating outward for longer than AX3 cells (which is consistent with observations of enhanced chemotaxis; Buczynski et al., 1997). After another hour a few small aggregates were observed in the double-knockout cells. After 45 hours AX3 cells stopped migration and started to aggregate and form fruiting bodies. Interestingly, a small percentage of pik1∆2∆ cells in both folate and cAMP gradients migrated much further than the majority of the population, again suggesting that aggregation rather than motility was impaired.
PI3K-II function revealed by gene knockout in Dictyostelium Therefore, there was no evidence of defects in the ability of pik1∆2∆ cells to generate gradients in cAMP and folate, indicating that folate deaminase and phosphodiesterase activities were probably not impaired. In addition, the doubleknockout cells were capable of movement along the gradient, indicating they were capable of detecting the gradients and moving accordingly. Nonetheless, the double-knockout mutant lacked the ability to form mounds, which could be explained by a defect in cAMP-induced cAMP secretion. A
Fig. 2. Lack of stream formation with double-knockout cells and complementation with DdPIK2. Confluent pik1∆2∆/pPIK2 (A) and pik1∆2∆ (B) cells were starved by substituting 12 mM Na/K-PO4 buffer for HL5 medium. After 10-14 hours of starvation, the complemented strain (C and D) and the wild type (not shown) formed long aggregation streams, with the aggregation center shown in C and the surrounding area shown in D, whereas the doubleknockout strains did not form aggregation streams (E). After 18 hours most of the complemented cells formed large aggregates; one of the smaller aggregates is shown in (F). In contrast, the doubleknockout cells formed numerous but very small aggregates after 2 to 3 days of starvation (G). ×20 (A,B), ×10(C-G).
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defect in cAMP-mediated aggregation in combination with normal motility could also explain why pik1∆2∆ forms shallow and diffuse plaques, and results in low fruiting-body density on a bacterial lawn (Zhou et al., 1995). However, cAMP levels, measured in starved cells by radioimmunoassay, were not different in wild-type and double-knockout cells (Fig. 4). Hence, differences in cAMP secretion do not appear to be responsible for the observed differences in stream formation.
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Fig. 3. The double-knockout cells are motile but do not aggregate into mounds. Dictyostelium cells were starved for 6 hours, pelleted and deposited on a thin layer of agarose with (bottom panel) or without (top panel) cAMP (20 mM). Pictures were taken 3 hours after cells were deposited. The larger diameter of the circle of complemented cells is probably not due to faster motility, but to higher initial cell number. This experiment was repeated three times with identical results.
The double-knockout cells have severe defects in pinocytosis The double-knockout cells grew more slowly compared to isogenic strains of single knockouts and wild-type strains in axenic growth medium. Furthermore, they did not grow in shaking culture, implicating defects in pinocytosis, the major means by which axenic strains obtain nutrients. We therefore analyzed pinocytosis in the knockout, wild-type and complemented strains. Interestingly, pik1∆2∆ had a very slow rate of pinocytosis (Fig. 5A). Pinocytosis of a liquid marker can be modeled by the following equation: 5
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Fig. 4. Comparison of cAMP production in wild-type, complemented and double-knockout cells. Cells were starved for 5 hours in phosphate buffer, placed on ice for 1 hour prior to stimulation with 10 µM deoxy cAMP and cAMP production was measured as described in Materials and methods. Wild type (n=2) (j); pik1∆2∆ (n=4) (s); pik1∆2∆/pPIK2 (n=2) (m). Data show a time course of cAMP production and are the means of 2-4 experiments. Error bars represent s.e.m. for data points where n>2.
V = k/j(1−e−jt) + B,
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where k is the influx rate, j is the efflux rate and B is the base line that reflects nonspecific binding of liquid marker and the natural fluorescence of the cell. The amount of the fluid marker, V, in the cell is a function of time, t. We define the equilibrium value of V as pinocytosis capacity (Vo), and Vo=k/j. By applying equation 1 to the experimental data, we obtained pinocytosis parameters (Fig. 5A, box). All pinocytosis parameters were reduced in pik1∆2∆, which only had 5% of the influx rate, 29% of the efflux rate, 17% of the pinocytosis capacity and 29% of the baseline levels of those of AX3. Part of the reduction in the magnitude of the pinocytosis parameters was due to the smaller cell size of pik1∆2∆. If we corrected for the reduction in pinocytosis that would result from the smaller surface area of smaller cells, and assume that pik1∆2∆ cells have one third of the cell surface area of the wild type (this is an underestimate), then the influx rate and pinocytosis capacity (which is proportional to the volume of the cell) would still be substantially lower than that of AX3 while the efflux rate would be very close to that of AX3. The parameters for the complemented cells were very close to those of the wild type. Altered phagocytosis of pik1∆2∆ We did not expect phagocytosis of pik1∆2∆ to be affected as drastically as pinocytosis because the cells still form plaques on a bacterial lawn, albeit at slower rates. Phagocytosis was assessed by two assays in shaking culture: (1) uptake of fluorescently labeled bacteria or (2) reduction in optical density at 590 nm (OD590) in co-cultures of bacteria and Dictyostelium cells. The ingestion rate of pik1∆2∆ assayed as uptake of fluorescently labeled bacteria was slightly slower than that of AX3 or pik1∆2∆/pPIK2 (Fig. 5B, squares). After approximately 30 minutes all three strains reached plateau values that varied according to cell size. The size of the cells in the different strains varied such that AX3>pik1∆2∆/pPIK2>pik1∆2∆. Ingestion of bacteria by Dictyostelium was clearly altered in the doubleknockout strain when analyzed by the reduction in optical density. All three strains were inoculated to the same initial OD590; however, pik1∆2∆ cells were smaller, permitting a higher density (3 times more cells per ml) (Fig. 5C, lower graph). After Dictyostelium cells were mixed with bacterial cells, AX3 and complemented cells immediately began to consume bacteria
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(Fig. 6C, upper graph), which was reflected as an immediate drop in OD590. In contrast, pik1∆2∆ had an initial increase in OD590, which only dropped below the initial value after 3 hours (Fig. 5C, upper graph). The initial increase of OD590 could not be explained by an increase in pik1∆2∆ cell numbers because that actually dropped during the experiment (Fig. 5C, lower graph). Therefore, these data indicate that the inability of the double-knockout cells to grow in shaking culture may not be caused by defects in nutrient uptake alone. Although wild type and complemented strains both had a doubling time of about 3 hours, AX3 cells were more numerous, whereas the cells of the complemented strain tended to be larger. The defect in phagocytosis in pik1∆2∆ was most dramatic when comparing phagocytosis of autoclaved bacteria. Autoclaved bacteria have denatured proteins and therefore have lost metabolic activities such as the production of folate that may be used by Dictyostelium cells as signals. The knockout mutant consumed autoclaved bacteria more slowly than AX3 (Fig. 5D) or pik1∆2∆/pPIK2 (data not shown). In summary, pik1∆2∆ were unable to phagocytose autoclaved bacteria and apparent phagocytosis of live bacteria occurred only after a prolonged delay. The data suggest that pik1∆2∆ can acquire nutrients by engulfing and digesting live, but not autoclaved, bacteria. Therefore the inability to divide in shaking culture cannot be explained by lack of nutrient uptake.
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Fig. 5. The double-knockout cells are defective in endocytosis. (A) Defects in pinocytosis as measured by liquid uptake as a function of time. The solid lines show calculated values based on parameters (defined in the text), as shown in the box below and equation 1. Data for the complemented cells was nearly identical to the wild type (data not shown). Box: Pinocytosis parameters of different strains. Values are for 109 cells. Units are k, ml min−1; j, ml min−1; Vo, ml; B, ml. Data shown are from one representative experiment, which was repeated three times. The altered phagocytosis behavior of the double-knockout cells is shown in B-D. (B) Phagocytosis of fluorescently labeled bacteria. These time courses were repeated 3 times using slightly different time points. Averaged percentage difference from wild type at 30 and 50 minutes (means±s.e.m.): pik1∆2∆/pPIK2, 30 minutes: 105±20%; pik1∆2∆/pPIK2, 50 minutes: 108±21.9%; pik1∆2∆, 30 minutes: 53±11.6%*; pik1∆2∆, 50 minutes: 46±9.6%**; *P
