Department of Biological Sciences, 132 Long Hall, Clemson University, Clemson, SC 29634, USA
Correspondence
Lesly A. Temesvari
LTEMESV{at}clemson.edu
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ABSTRACT |
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INTRODUCTION |
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Dictyostelium discoideum offers numerous advantages as an experimental model system in which to study the role of cellcell adhesion, developmentally regulated secretion, and actin in developmental processes. It possesses a simple and well-defined life cycle consisting of a vegetative phase and a developmental phase (reviewed by Loomis, 1975). During the vegetative phase, the organism survives as a free-living amoeba in the presence of sufficient nutrients. Starvation results in a switch to development during which individual cells emit pulses of cAMP to which other cells respond chemotactically. Streams of cells interact through cellcell adhesions, and form a multicellular aggregate of about 105 cells. The aggregate then undergoes a programme of cell-type-specific gene expression and cytodifferentiation to produce an intermediate slug, and finally, a fruiting body that contains a stalk supporting environmentally stable spores.
Several of the CAMs that participate in Dictyostelium development have been described (reviewed by Coates & Harwood, 2001). Dictyostelium cells form their initial contacts through interactions of a plasma membrane-associated Ca2+-dependent cadherin-like protein known as gp24 (DdCAD-1) (Yang et al., 1997
). In early development this CAM is transported from the cytosol to the external surface of the plasma membrane and a small fraction of this CAM is released into the extracellular milieu. The second and third adhesion molecules to participate in cellcell interactions during development are gp80 and gp150, respectively (Faix et al., 1992
; Wang et al., 2000
). Following the incorporation of gp80 and gp150 at cellcell contact sites, filamentous actin (F-actin) is recruited to the contact site, whereas gp24 is removed from the contact site. The loss of gp24 is postulated to be necessary to weaken the adhesive forces between cells to facilitate streaming; however, the manner by which it is removed is unknown.
Regulated secretion is also characteristic of D. discoideum development. When cells reach high density and during early development, D. discoideum amoebae secrete lysosomal hydrolases (Cardelli, 1993; Temesvari et al., 1996
), cell-density sensing factor (CMF) (Gomer et al., 1991
; Jain et al., 1992
; Yuen et al., 1995
) and cell-counting factor (CF) (Brock & Gomer, 1999
; Roisin-Bouffay et al., 2000
; Brock et al., 2002
; Jang et al., 2002
; Tang et al., 2002
). CF is a 450 kDa protein complex that can regulate aggregate size during development. Countin, a subunit of CF, has been shown to negatively regulate adhesion by regulating the expression of CAMs (Roisin-Bouffay et al., 2000
). For example, in mutants (smlA) which oversecrete countin, there is decreased adhesion and delayed expression of gp24, whereas in mutants in which the gene for countin is disrupted (countin) there is increased adhesion and expression of gp24 (Roisin-Bouffay et al., 2000
). Like countin, CF45, another subunit of CF, also participates in group size regulation in Dictyostelium. Finally, at later stages in development (
20 h), secretion of the contents of prespore vesicles is necessary for the formation of the spore coat (reviewed by Srinivasan et al., 2000
).
While much is known about the types of proteins that become part of adhesions or that are secreted during Dictyostelium development, little is known about how these molecules are delivered to the cell surface and/or secreted. Presumably, the movement of these proteins relies on vesicle trafficking, suggesting that the Ras-related Rab GTPases, master controllers of this process (reviewed by Pfeffer, 2001; Segev, 2001
), may be involved. Rabs share a number of homologous domains, including four GTP-interacting domains (G1G4), five Rab-specific functional domains (F1F5) and four subfamily-specific domains (SF1SF4) (reviewed by Pereira-Leal & Seabra, 2000
; Stenmark & Olkkonen, 2001
). In general, phylogenetic analyses of the F1 and SF1SF4 domains have revealed the existence of ten subfamilies of Rab GTPases. Rabs cycle between an active GTP-bound state and an inactive GDP-bound form and associate with transport vesicles through C-terminal lipid modifications (Schafer & Rine, 1992
). After vesicle docking at target membranes, GTP hydrolysis, via the intrinsic GTPase activity of the Rab, converts the Rab to its GDP-bound form. Rab GTPase activity is regulated by GTPase-activating proteins (GAPs) and the activation of Rabs by nucleotide exchange relies on guanine nucleotide exchange factors (GEFs). Rabs are proposed to interact with GAPs and GEFs through their effector (F1) domain.
Rab8-related GTPases represent a subfamily of Rabs that include mammalian Rab8 (Peranen et al., 1996; Imamura et al., 1998
; Hattula et al., 2002
; Lau & Mruk, 2003
), Rab13 (Zahraoui et al., 1994
; Sheth et al., 2000
; Marzesco et al., 2002
), Rab10 (Chen et al., 1993
), Rab3A (Vadlamudi et al., 2000
) and yeast Sec4p (Guo et al., 1999
). These Rabs participate in cellular functions that may be vital to development as they regulate polarized secretion, actin cytoskeletal dynamics, and cellcell adhesion. In support of this, mammalian Rab13 has been shown to participate in the maturation of epithelial tight junctions during embryogenesis (Sheth et al., 2000
). To further our understanding of the molecular factors that govern cellcell adhesion, the actin cytoskeleton and regulated secretion in a developmental context, we have characterized a Rab8-like protein of Dictyostelium, Sas1. This protein, first identified by Saxe & Kimmel (1990)
, is present in vegetative cells; however, the level of transcript increases immediately at the onset of development, reaching a maximum level at 15 h into the developmental programme. This pattern of gene expression suggests that this protein plays an important role in early development and we demonstrate that, like other Rab8-related proteins, Sas1 may regulate the arrangement of actin, cellcell adhesion and secretion. As a control, we also examined a second highly related Rab8-like protein of Dictyostelium, Sas2 (Saxe & Kimmel, 1990
). The expression pattern of Sas2, characterized by low levels of transcript in vegetative cells and an increase in transcript beginning only after 15 h into development, suggests that it is a likely candidate for the regulation of cellular processes important at later stages in development.
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METHODS |
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Strains and culture conditions.
D. discoideum wild-type (Ax2) and mutant cell lines (see below) were grown axenically in HL5 medium as described previously (Temesvari et al., 1996). Transformed cell lines expressing mutant Sas proteins (see below) were maintained by G418 selection (20 µg ml1) and folic acid (1x103 M) repression. Prior to all experimentation, mutant cells were cultured for 72 h in the absence of folic acid to induce expression of the mutant Sas proteins from the folate-repressible discoidin I promoter. For development, cells were resuspended at a concentration of 5·5x105 to 2·5x106 cells ml1 in developmental buffer (DB) (5 mM Na2HPO4.7H2O, 5 mM KH2PO4, 2 mM MgSO4, 0·2 mM CaCl2, pH 6·2) or plated at a concentration of 1x106 cells cm2 on DB plates containing 2 % (w/v) agar and incubated at 22 °C in the dark in a humid chamber.
Antibodies.
Antibodies recognizing GFP were obtained from Zymed. Polyclonal antibodies recognizing the adhesion molecule, gp24 (gift of Dr C. H. Siu, University of Toronto, Toronto, Ontario, Canada) are described elsewhere (Brar & Siu, 1993). Polyclonal antibodies recognizing subunits of CF (countin, CF45) (gift of Dr R. H. Gomer, Rice University, Houston, TX, USA) are described elsewhere (Brock & Gomer, 1999
; Brock et al., 2003
). Sas1-specific polyclonal antibodies were produced by immunizing rabbits (Zymed) with a synthetic peptide corresponding to amino acids 185200 in the C-terminal divergent region of Sas1 (dotted underlined sequence, Fig. 1
). Antibodies were purified by affinity chromatography (Zymed) using an affinity column consisting of immobilized Sas1 peptide. The specificity of the antibodies was assessed by Western blotting (see below).
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To assess the extracellular levels of gp24, CF45, or countin, exponential-phase Dictyostelium cells were resuspended at 5x106 cells ml1 in HL5 nutrient medium or DB and shaken at 150 r.p.m. for 18 h or for 2 h, respectively. Cells and conditioned medium or DB were separated by centrifugation at 16 000 g for 4 min at 22 °C to pellet the cells. The supernatant was supplemented with a cocktail of protease inhibitors (1 mM PMSF, 1 µg pepstatin ml1, 10 µg leupeptin ml1) and proteins >10 kDa were concentrated (approx. 10-fold) by centrifugation (2000 g) using a centrifugal filter device (10 kDa cutoff, Millipore). After centrifugation, the entire volume was mixed with an equal volume of 4x LDS sample buffer (Invitrogen) and 2-mercaptoethanol (10 % v/v), heated at 70 °C for 10 min and subjected to SDS-PAGE as described above. In some instances, to verify equal loading of samples, the gels were silver stained using the GelCode SilverSNAP stain kit (Pierce) according to the manufacturer's protocol. Alternatively, resolved proteins were blotted and decorated with antibodies specific for gp24, CF45 or countin as described above. The dilution used for both the anti-countin and anti-CF45 antibodies was 1 : 3000.
Mutagenesis of the Sas cDNAs and transfection of Dictyostelium.
To generate Dictyostelium mutants that conditionally overexpress mutant versions of Sas1, PCR-based site-directed mutagenesis was performed using the QuickChange Kit (Stratagene) according to the manufacturer's instructions. Site-directed mutagenesis was used to change an encoded glutamine residue (Q) to a leucine residue (L) at amino acid position 74 (region G2, Fig. 1) to generate a Sas1Q74L mutant. Alternatively, an encoded asparagine residue (N) was changed to an isoleucine residue (I) at amino acid position 128 (region G3, Fig. 1
) to generate a Sas1N128I mutant. The glutamine residue participates in GTP hydrolysis and the asparagine residue participates in nucleotide binding. Comparable mutations at equivalent glutamine or asparagine residues in other small-molecular-mass GTP-binding proteins have resulted in the formation of proteins that function in a constitutively activated or dominant negative manner, respectively. As a control, corresponding mutations were also made for Sas2.
Wild-type and mutated cDNAs were subcloned behind and in-frame with a DNA element encoding the GFP in the pDJS Dictyostelium expression vector (gift of Dr J. A. Cardelli, LSU Health Sciences Center, Shreveport, LA, USA). The expression vector confers neomycin (G418) resistance to transfectants and the expression of heterologous proteins is controlled by a folate-repressible discoidin I promoter. Parental Ax2 Dictyostelium cells were transformed with the Sas-containing expression vectors or with the pDJS expression vector alone by electroporation as described by Kuspa & Loomis (1992). After transfection, G418-resistant clones were sorted by fluorescence-associated cell sorting (FACS) and cloned by limiting dilution.
Microscopy.
In individual cells, F-actin was stained using Alexa Fluor 594 (red)-conjugated phalloidin (Molecular Probes) according to the manufacturer's protocol. Stained cells were mounted in glycerol/PBS (1 : 1) and observed using a Carl Zeiss LSM 510 confocal microscope. Measurements of aggregate size were obtained using the LSM 5.1 Image Browser software (Carl Zeiss). Images of fruiting bodies were obtained using a stereomicroscope (Wild Heerbrugg) and a Kodak DC120 digital camera.
Measurement of actin.
Actin was measured according to the protocol of Gerald et al. (1998) with modifications. Exponential-phase cells (3x106) were collected by centrifugation and the pellet was resuspended in 500 µl PBS. Twenty percent of the cells was lysed in 0·5 % (v/v) Triton-X-100 and utilized for measurement of protein with the BCA Protein Assay Kit (Pierce). The remaining cells were rotated for 1 h at room temperature in the dark in actin buffer (20 mM KH2PO4, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6·8) supplemented with 4 % (v/v) paraformaldehyde, 0·1 % (v/v) Triton-X-100 and 0·2 µM Alexa Fluor 594-conjugated phalloidin (actin stain). The stained cells were centrifuged at 16 000 g for 5 min, resuspended in 500 µl methanol, and rotated overnight in the dark at 4 °C to extract the stain. The extracted cells were then centrifuged and fluorescence of the supernatant was measured at an excitation wavelength of 590 nm and an emission of 635 nm using a FLx800 spectrofluorimeter (BioTek Instruments). Data are reported as relative fluorescence (µg protein)1.
Measurement of adhesivity.
Adhesion was measured as described previously (Desbarats et al., 1994). To measure adhesion during development, cells were harvested by centrifugation and resuspended at a concentration of 5·5x105 cells ml1 in 200 µl DB. To measure adhesion during vegetative growth, cells were harvested by centrifugation and resuspended at a concentration of 2·5x106 cells ml1 in 200 µl HL5 medium. This higher cell density was chosen to adjust for the lower adhesiveness that is characteristic of vegetative cells (Gao et al., 2002
). In some instances, to assess the contribution of Ca2+-dependent CAMs, EDTA was added to a final concentration of 10 mM as described by Roisin-Bouffay et al. (2000)
. Resuspended cells were rotated vertically for 10 min at room temperature after which adhesion was estimated by counting the number of single cells and the number of aggregated cells, including doublets, using a haemocytometer. Adhesion is reported as the percentage of cells that were adhering to other cells.
Measurement of secretion of acid phosphatase.
Measurement of acid phosphatase secretion was performed as described previously (Temesvari et al., 1996).
Statistical analysis.
All values are given as a mean±SD. Statistical analyses were performed using GraphPad Instat V.3 with One Way ANOVA and a Tukey-Multiple Comparison test. P values less than 0·01 were considered highly statistically significant and P values between 0·01 and 0·05 were considered statistically significant.
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RESULTS |
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To assess the expression of mutant Sas proteins, polyclonal antibodies were generated using a peptide corresponding to the C-terminal divergent region of Sas1 (dotted underline, Fig. 1). Affinity purified antiserum recognized a band on Western blots of whole-cell lysates consistent with the predicted molecular mass of the protein (Fig. 2a
, lane 1). A similar sized band was not recognized by pre-immune serum nor by secondary antibody (Fig. 2a
, lanes 2 and 3, respectively). Occasionally, an unidentified protein species of approximately 40 kDa was recognized by immune and pre-immune serum (Fig. 2a
, lanes 1 and 2). As an additional control, the anti-Sas1 antibody was pre-incubated with Sas1 and Sas2 synthetic peptides prior to Western blot analyses. Binding of antibody to the putative Sas1 protein species on Western blots was abolished by pre-incubating the antibody with the Sas1 synthetic peptide (Fig. 2b
, lane 2), but not with a Sas2 synthetic peptide (Fig. 2b
, lane 3).
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Expression of Sas1 mutant proteins alter the actin cytoskeleton
Rab8-like proteins have been shown to regulate the formation of membrane extensions by altering the actin cytoskeleton (Peranen et al., 1996). Therefore, to explore the functional relatedness of Sas1 to Rab8-like proteins we examined these features in the mutant cell lines. Vegetative Dictyostelium cells are generally amoeboid in shape and possess a cortical actin cytoskeleton localized at the cell periphery. Overexpression of GFP-alone (Fig. 3A
) or Sas1WT (Fig. 3B
) had no effect on cell shape or the arrangement of actin as evidenced by F-actin staining. While the plasma membrane of cells expressing Sas2WT or Sas2Q74L (Fig. 3C, D
) appeared slightly more ruffled than control cells, the F-actin cytoskeleton was enriched in the cortical regions of the cell. Sas1N128I mutants, although appearing slightly less amoeboid, demonstrated little apparent change in the localization of the actin cytoskeleton (Fig. 3E
H). In contrast, expression of Sas1Q74L induced the formation of membrane extensions that were concentrated in one or several locations on the cells (Fig. 3IL
). The formation of these extensions was accompanied by a change in the localization of actin whereby most of the F-actin in the cell was now concentrated in the polarized cellular protrusions. Although the arrangement of actin was altered in the Sas1Q74L cell line, the level of F-actin was relatively constant among the cell lines (Fig. 3M
). Together, these data suggest that, like mammalian Rab8, Sas1 may regulate the arrangement of the actin cytoskeleton and the formation of cellular extensions.
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Importantly, there were no detectable differences in adhesivity among the cell lines in the presence of EDTA (Fig. 6b), suggesting that Sas1 may regulate cellcell adhesions via a Ca2+-dependent CAM such as gp24. We therefore performed a Western blot analysis of cell lysates from control and mutant cells using antibodies specific for gp24. Since gp24 may also be released into the extracellular milieu, we also performed a similar Western blot analysis of conditioned medium. Consistent with the observed alterations in adhesivity, Western blot analysis demonstrated that total (cell-associated and extracellular) gp24 was less abundant for Sas1Q74L cells and more abundant for Sas1N128I cells as compared to the other cell lines (Fig. 6c
).
Expression of Sas1Q74L alters the extracellular level of countin
Given that the Sas1N128I mutant had a gain of function adhesion phenotype, and that the Sas1Q74L mutant had a loss of function adhesion phenotype, we hypothesized that the role of Sas1 in adhesion was indirect. Several members of the Rab8 family of GTPases, including mammalian Rab8 (Chen et al., 2001), Rab3A (Vadlamudi et al., 2000
) and Saccharomyces Sec4p (Guo et al., 1999
), have been shown to regulate secretion. Therefore, one possible explanation may be that the Sas1Q74L cell line oversecretes a signalling molecule or molecules that in turn regulate adhesion. By deduction, the Sas1N128I cell line would undersecrete such signalling molecules. Given the increased expression of Sas1 early in development, it is conceivable that this Rab may regulate secretory events that are important during this phase of the life cycle. One candidate signalling molecule that would account for the observed phenotypes would be CF, which is secreted when cells reach high density and during starvation. One subunit, countin, negatively regulates adhesion by regulating the expression of gp24 and other CAMs (Roisin-Bouffay et al., 2000
).
To test the possibility that Sas1 regulates the extracellular levels of countin, control and mutant cells were incubated at high density in nutrient medium or starved in DB and the level of extracellular countin was examined by Western blot analysis. In nutrient medium, there were minimal detectable differences in the level of extracellular countin among the cell lines (Fig. 7a). However, during starvation the level of extracellular countin was higher for the Sas1Q74L cell line as compared to the other cell lines (Fig. 7b
). Scanning densitometry indicated that the level of extracellular countin was 1·7-fold higher for the Sas1Q74L cell line as compared to control cells. This suggests that Sas1 may regulate the release of countin during starvation and the increased level of extracellular countin may account for the decrease in the level of the gp24 observed for this cell line.
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Expression of Sas1Q74L alters the extracellular level of acid phosphatase
Given the role of Rab8-like GTPases in secretion (Chen et al., 2001; Vadlamudi et al., 2000
; Guo et al., 1999
), we also performed standard secretion assays to measure the rate of secretion of a lysosomal hydrolase, acid phosphatase. Exponentially growing cells were harvested and resuspended in growth medium, and at 2 h the activity of acid phosphatase was measured in both cells and the medium (Fig. 8
). In growth medium, acid phosphatase was secreted from all of the cell lines to the same extent (approx. 25·7 % of the total enzyme activity was found in the extracellular medium at 2 h) (Fig. 8
, open bars). In addition to initiating development, starvation also induces slight increases in the secretion of lysosomal hydrolases (Cardelli, 1993
; Temesvari et al., 1996
). This response was examined in the Sas mutants by performing standard secretion assays, as described above, using cells that had been resuspended in DB (Fig. 8
, closed bars). All of the cell lines responded to starvation by slight increases in the secretion of acid phosphatase. However, the response was significantly pronounced in the Sas1Q74L-expressing mutant line which displayed a level of secretion that was 6·3-fold higher than that of the Sas1Q74L-expressing mutant in nutrient medium and 2·3-fold higher than that of the GFP-control in starvation conditions. This suggests that Sas1 may play a role in secretion during development.
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DISCUSSION |
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Regulation of tissue size is critical during development and one of the ways in which Dictyostelium cells sense group size is through an extracellular protein complex, namely counting factor, CF (Brock & Gomer, 1999; Roisin-Bouffay et al., 2000
; Brock et al., 2002
, 2003
; Jang et al., 2002
; Tang et al., 2002
). Since Rab8-related proteins regulate secretory events in a variety of cell types (Huber et al., 1993
, 1995
; Guo et al., 1999
; Chen et al., 2001
), it is not surprising that Sas1, a developmentally regulated Rab8-like protein, may control similar processes in Dictyostelium. Interestingly, the extracellular levels of CF45 were not altered for the Sas1Q74L mutants, suggesting that the mutant does not have a generalized secretion defect and that the release of the various CF subunits may rely on different mechanisms. This is consistent with a previous report (Brock et al., 2003
) that demonstrates that the subunits of CF are not secreted as a stoichiometric complex.
Although the Sas1N128I cells formed large aggregates, the aggregates became fragmented at later developmental time points. It has been shown that overexpression of the CAM, gp80, results in aggregates that are less flexible and unable to withstand shearing forces generated during coordinated cell motility (Faix et al., 1992). This situation leads to dissociation of aggregates and the formation of smaller fruiting bodies. Therefore, the inability of the Sas1N128I cell line to remain adherent during development may be due to the inappropriately high levels of gp24; however, we cannot rule out the possibility that the levels of other CAMs are also increased in this cell line. Interestingly, several of the Rab8-related proteins have also been implicated in adhesion (Sheth et al., 2000
; Marzesco et al., 2002
; Lau & Mruk, 2003
).
Cells in which the gene encoding gp24 is disrupted are able to complete development; however, these mutants display delayed culmination and abnormal slug and fruiting body morphology (Wong et al., 2002). In contrast, blocking gp24-mediated cellcell interactions using anti-gp24 antibodies or purified gp24 completely inhibits aggregation (Knecht et al., 1987
; Brar & Siu, 1993
; Wong et al., 1996
). This raises the question of why the developmental outcome of loss of gp24 through genetic disruption is different from that of functional loss through interference. It is possible that other uncharacterized CAMs compensate for gp24 in the null cell line. In support of this, gp24 null cells exhibit precocious expression of gp80 (Wong et al., 2002
). Moreover, disruption of gp24 resulted only in a 50 % reduction in EDTA-sensitive adhesions, indicating the existence of additional Ca2+-dependent CAMs (Wong et al., 2002
). Surprisingly, although significantly reduced in gp24, Sas1Q74L-expressing cells displayed a non-aggregative phenotype similar to that of gp24-blocked. The severity of this defect may suggest that multiple CAMs may be affected in the Sas1Q74L-expressing mutant.
It is indeed interesting that, in addition to their role in vesicle trafficking, Rab8-like proteins regulate the actin cytoskeleton and filopodia formation (Peranen et al., 1996; Imamura et al., 1998
; Hattula et al., 2002
). During D. discoideum development, membranes of streaming cells first make contact through filopodia that are rich in gp24 (Sesaki & Siu, 1996
). It is intriguing that a developmentally regulated Rab8-like protein, Sas1, might regulate both the formation of these filopodia and the level of gp24. Since the observed changes in gp24 are not sufficient to explain the phenotype of the Sas1Q74L-expressing mutant, the severity of the aggregation defect observed may be the result of combined defects in filopodia formation, secretion of counting factor, and levels of gp24.
This is the first report describing the function of a developmentally regulated Rab8-like protein of Dictyostelium. During development, secretion, actin cytoskeletal changes and cellcell adhesion are important in controlling multicellular structure formation. These studies identify for the first time a Rab protein that may regulate several of these processes simultaneously. Identification of Sas1-interacting proteins and secreted proteins from Sas1 mutants will provide further insight into the mechanism by which Sas1 and other Rab8-related proteins function.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brar, S. K. & Siu, C. H. (1993). Characterization of the cell adhesion molecule gp24 in Dictyostelium discoideum: mediation of cell-cell adhesion via a Ca2+-dependent mechanism. J Biol Chem 268, 2490224909.
Brock, D. A. & Gomer, R. H. (1999). A cell-counting factor regulating structure size in Dictyostelium. Genes Dev 13, 19601969.
Brock, D. A., Hatton, R. D., Giurgiutiu, D. V., Scott, B., Ammann, R. & Gomer, R. H. (2002). The different components of a multisubunit cell number-counting factor have both unique and overlapping functions. Development 129, 36573668.
Brock, D. A., Hatton, R. D., Giurgiutiu, D. V., Scott, B., Jang, W., Ammann, R. & Gomer, R. H. (2003). CF45-1, a secreted protein which participates in Dictyostelium group size regulation. Eukaryot Cell 2, 788797.
Buckley, C. D., Rainger, G. E., Bradfield, P. F., Nash, G. B. & Simmons, D. L. (1998). Cell adhesion: more than just glue. Mol Membr Biol 15, 167176.[Medline]
Cardelli, J. A. (1993). Regulation of lysosomal trafficking and function during growth and development of Dictyostelium discoideum. In Advances in Cell and Molecular Biology of Membranes, vol. 1, pp. 341390. Edited by B. Storrie & R. Murphy. Greenwich, CT: Jai Press.
Chen, S., Liang, M. C., Chia, J. N., Ngsee, J. K. & Ting, A. E. (2001). Rab8b and its interacting partner TRIP8b are involved in regulated secretion in AtT20 cells. J Biol Chem 276, 1320913216.
Chen, Y. T., Holcomb, C. & Moore, H. P. (1993). Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag. Proc Natl Acad Sci U S A 90, 65086512.[Abstract]
Coates, J. C. & Harwood, A. J. (2001). Cell-cell adhesion and signal transduction during Dictyostelium development. J Cell Sci 114, 43494358.[Medline]
Desbarats, L., Brar, S. K. & Siu, C. H. (1994). Involvement of cell-cell adhesion in the expression of the cell cohesion molecule gp80 in Dictyostelium discoideum. J Cell Sci 107, 17051712.
Faix, J., Gerisch, G. & Noegel, A. A. (1992). Overexpression of the csA cell adhesion molecule under its own cAMP-regulated promoter impairs morphogenesis in Dictyostelium. J Cell Sci 102, 203214.[Abstract]
Gao, T., Ehrenman, K., Tang, L., Leippe, M., Brock, D. A. & Gomer, R. H. (2002). Cells respond to and bind countin, a component of a multisubunit cell number counting factor. J Biol Chem 277, 3259632605.
Gerald, N., Dai, J., Ting-Beall, H. P. & De Lozanne, A. (1998). A role for Dictyostelium racE in cortical tension and cleavage furrow progression. J Cell Biol 141, 483492.
Gomer, R. H., Yuen, I. S. & Firtel, R. A. (1991). A secreted 80x103 Mr protein mediates sensing of cell density and the onset of development in Dictyostelium. Development 112, 269278.[Abstract]
Guo, W., Roth, D., Walch-Solimena, C. & Novick, P. (1999). The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J 18, 10711080.
Hattula, K., Furuhjelm, J., Arffman, A. & Peranen, J. (2002). A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. Mol Biol Cell 13, 32683280.
Huber, L. A., Pimplikar, S., Parton, R. G., Virta, H., Zerial, M. & Simons, K. (1993). Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol 123, 3545.[Abstract]
Huber, L. A., Dupree, P. & Dotti, C. G. (1995). A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons. Mol Cell Biol 15, 918924.[Abstract]
Imamura, H., Takaishi, K., Nakano, K., Kodama, A., Oishi, H., Shiozaki, H., Monden, M., Sasaki, T. & Takai, Y. (1998). Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells. Mol Biol Cell 9, 25612575.
Jain, R., Yuen, I. S., Taphouse, C. R. & Gomer, R. H. (1992). A density-sensing factor controls development in Dictyostelium. Genes Dev 6, 390400.[Abstract]
Jang, W., Chiem, B. & Gomer, R. H. (2002). A secreted cell number counting factor represses intracellular glucose levels to regulate group size in Dictyostelium. J Biol Chem 277, 3920239208.
Knecht, D. A., Fuller, D. L. & Loomis, W. F. (1987). Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum. Dev Biol 121, 277283.[Medline]
Kuspa, A. & Loomis, W. F. (1992). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A 89, 88038807.[Abstract]
Lau, A. S. & Mruk, D. D. (2003). Rab8B GTPase and junction dynamics in the testis. Endocrinology 144, 15491563.
Loomis, W. F. (1975). Dictyostelium discoideum: a Developmental System. New York: Academic Press.
Marzesco, A. M., Dunia, I., Pandjaitan, R., Recouvreur, M., Dauzonne, D., Benedetti, E. L., Louvard, D. & Zahraoui, A. (2002). The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells. Mol Biol Cell 13, 18191831.
Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J. & Wittinghofer, A. (1989). Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341, 209214.[CrossRef][Medline]
Peranen, J., Auvinen, P., Virta, H., Wepf, R. & Simons, K. (1996). Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J Cell Biol 135, 153167.[Abstract]
Pereira-Leal, J. B. & Seabra, M. C. (2000). The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J Mol Biol 301, 10771087.[CrossRef][Medline]
Pfeffer, S. R. (2001). Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol 11, 487491.[CrossRef][Medline]
Roisin-Bouffay, C., Jang, W., Caprette, D. R. & Gomer, R. H. (2000). A precise group size in Dictyostelium is generated by a cell-counting factor modulating cell-cell adhesion. Mol Cell 6, 953959.[Medline]
Saxe, S. A. & Kimmel, A. R. (1990). Sas1 and Sas2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae. Mol Cell Biol 10, 23672378.[Medline]
Schafer, W. R. & Rine, J. (1992). Protein prenylation: genes, enzymes, targets, and functions. Annu Rev Genet 26, 209237.[CrossRef][Medline]
Segev, N. (2001). Ypt and Rab GTPases: insight into functions through novel interactions. Curr Opin Cell Biol 13, 500511.[CrossRef][Medline]
Sesaki, H. & Siu, C. H. (1996). Novel redistribution of the Ca(2+)-dependent cell adhesion molecule DdCAD-1 during development of Dictyostelium discoideum. Dev Biol 177, 504516.[CrossRef][Medline]
Sheth, B., Fontaine, J. J., Ponza, E., McCallum, A., Page, A., Citi, S., Louvard, D., Zahraoui, A. & Fleming, T. P. (2000). Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction. Mech Dev 97, 93104.[CrossRef][Medline]
Srinivasan, S., Alexander, H. & Alexander, S. (2000). Crossing the finish line of development: regulated secretion of Dictyostelium proteins. Trends Cell Biol 10, 215219.[CrossRef][Medline]
Stenmark, H. & Olkkonen, V. M. (2001). The Rab GTPase family. Genome Biol 2, 3007.13007.7.
Tang, L., Gao, T., McCollum, C., Jang, W., Vicker, M. G., Ammann, R. R. & Gomer, R. H. (2002). A cell number-counting factor regulates the cytoskeleton and cell motility in Dictyostelium. Proc Natl Acad Sci U S A 99, 13711376.
Temesvari, L. A., Bush, J. M., Peterson, M. D., Novak, K. D., Titus, M. A. & Cardelli, J. A. (1996). Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants. J Cell Sci 109, 663673.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Vadlamudi, R. K., Wang, R. A., Talukder, A. H., Adam, L., Johnson, R. & Kumar, R. (2000). Evidence of Rab3A expression, regulation of vesicle trafficking, and cellular secretion in response to heregulin in mammary epithelial cells. Mol Cell Biol 20, 90929101.
Wang, J., Hou, L., Awrey, D., Loomis, W. F., Firtel, R. A. & Siu, C. H. (2000). The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development. Dev Biol 227, 734745.[CrossRef][Medline]
Welter, B. H., Laughlin, R. C. & Temesvari, L. A. (2002). Characterization of a Rab7-like GTPase, EhRab7: a marker for the early stages of endocytosis in Entamoeba histolytica. Mol Biochem Parasitol 121, 254264.[CrossRef][Medline]
Wong, E. F., Brar, S. K., Sesaki, H., Yang, C. & Siu, C. H. (1996). Molecular cloning and characterization of DdCAD-1, a Ca2+-dependent cell-cell adhesion molecule, in Dictyostelium discoideum. J Biol Chem 271, 1639916408.
Wong, E., Yang, C., Wang, J., Fuller, D., Loomis, W. F. & Siu, C. H. (2002). Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development. Development 129, 38393850.[Medline]
Yang, C., Brar, S. K., Desbarats, L. & Siu, C. H. (1997). Synthesis of the Ca(2+)-dependent cell adhesion molecule DdCAD-1 is regulated by multiple factors during Dictyostelium development. Differentiation 61, 275284.[CrossRef][Medline]
Yuen, I. S., Jain, R., Bishop, J. D., Lindsey, D. F., Deery, W. J., Van Haastert, P. J. & Gomer, R. H. (1995). A density-sensing factor regulates signal transduction in Dictyostelium. J Cell Biol 129, 12511262.[Abstract]
Zahraoui, A., Joberty, G., Arpin, M., Fontaine, J. J., Hellio, R., Tavitian, A. & Louvard, D. (1994). A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells. J Cell Biol 124, 101115.[Abstract]
Received 30 January 2004;
revised 28 April 2004;
accepted 7 May 2004.
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