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© The Rockefeller University Press, 0021-9525/2000//823 $5.00
The Journal of Cell Biology, Volume 150, Number 4, , 2000 823-838

Dynacortin, a Genetic Link between Equatorial Contractility and Global Shape Control Discovered by Library Complementation of a Dictyostelium discoideum Cytokinesis Mutant

^a Department of Biochemistry and Developmental Biology, Beckman Center, Stanford University, Stanford, California 94305-5307
Department of Biochemistry and Developmental Biology, Beckman Center, Rm. B-400, Stanford University, Stanford, CA 94305-5307.650-725-6044650-725-6376

drobinso{at}cmgm.stanford.edu

We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.

Key Words: cytoskeleton • cortexillin • cell cortex • cell protrusion • Rac small GTPase

© 2000 The Rockefeller University Press

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokinesis is the process by which a cell divides into two daughter cells. Over the past century, several studies have helped to uncover the cellular mechanisms of cytokinesis. The following general theme for cytokinesis has emerged from studies on organisms ranging from yeast and amoeboid cells to echinoderm eggs and mammalian cells. The astral and/or interdigitating microtubules from the mitotic spindle send a signal to the equatorial cortex, triggering its contraction. This creates a cortical tension gradient in which the peak of the tension occurs at the equator and the lowest point of tension occurs at the cell poles. This leads to the constriction of the cell at the equator, cleaving the cell into two daughter cells (for review see Rappaport 1996; Robinson and Spudich 2000).

However, until recently, the molecular basis of cytokinesis has remained largely elusive, due in large part to the transient nature of the process compared with the length of the cell cycle. The conventional nonmuscle myosin II (myo II) contributes substantially to the equatorial contractile force in a number of organisms (for example, DeLozanne and Spudich 1987; McCollum et al. 1995; Bezanilla et al. 1997; Kitayama et al. 1997), but it might not be the sole contributor to equatorial contractile force generation. In the cellular slime mold, Dictyostelium discoideum, myo II mutant cells are capable of cleaving into two cells if cells are grown on surfaces. In mammals, myosin Is of the brush border subfamily are also localized to the cleavage furrow (Breckler and Burnside 1994; Ruppert et al. 1995), and in Schizosaccharomyces pombe, two different conventional myo IIs can be found in the cleavage furrow (Bezanilla et al. 1997; Kitayama et al. 1997).

Nonmotor proteins have been shown to generate cortical tension and regulate cell shape. In D. discoideum, cortexillins encoded by the cortexillin I and II genes are required for cytokinesis (Faix et al. 1996). The cortexillins consist of an NH₂-terminal spectrin-family actin binding domain, a coiled-coil domain, and a COOH-terminal domain that has actin filament-bundling activity and a PIP₂-binding site. Cortexillin I localizes to the cleavage furrow and the COOH-terminal domain is sufficient for cortexillin I's function in cytokinesis (Weber et al. 1999). This COOH-terminal domain is also sufficient for actin filament bundling in vitro (Stock et al. 1999). Loss of cortexillin function leads to a reduction in cortical tension (Simson et al. 1998). D. discoideum coronin is an actin filament-crosslinking protein that is required to maintain cell shape and loss of it leads to defects in cytokinesis (deHostas et al. 1991, deHostas et al. 1993). Mutations in the RacE small GTPase gene lead to reduced general cortical tension and failure in cytokinesis, implicating a signaling cascade that modulates cortical tension (Larochelle et al. 1997; Gerald et al. 1998).

In recent years, many new proteins have been identified that are involved in or required for cytokinesis. Many of these proteins have been discovered in D. discoideum, yeast, Caenorhabditis elegans, and Drosophila melanogaster. Only the cellular slime mold, D. discoideum, offers development and metazoan-type cellular physiology, as well as good cytology for microscopy, molecular genetic methods including insertional mutagenesis, and the ability to produce preparative quantities of cells for biochemical analyses (for review see Kay and Williams 1999). However, complex genetic interactions between molecules are still difficult to probe. This is due in part to the lack of a meiotic phase in laboratory D. discoideum strains, which restricts the ability to map mutations by recombination and greatly inhibits the ability of using genetic recombination to identify epistatic relationships. To dissect further the molecular mechanisms of cytokinesis, we have developed library complementation and high copy suppression methods to perform interaction genetics in D. discoideum. This has allowed us to identify a novel gene, dynacortin, which plays a role in the function of a dynamic cell cortex and interacts genetically between regional and global cortical tension generating pathways that are necessary for efficient cleavage of the cell.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
D. discoideum Strains and Cell Culture
For an isogenic parental wild-type strain for the mutagenesis experiments, a single subclone (ORF+7-3; HS1000) of ORF+ cells (Manstein et al. 1995) was isolated (Table ). The ORF+ cell line was chosen because it contains the pRep open reading frame (ORF), which is stably integrated in the genome. The pRep ORF is required for the replication of Ddp2 replication origins in D. discoideum, and can function in trans (Chang et al. 1990; Slade et al. 1990). Having the ORF integrated in the genome allows Ddp2-based episomal vectors to be transformed into D. discoideum cells without the use of the helper pRep plasmid.

D. discoideum ORF+7-3–derived strains were grown in Hans' enriched HL-5 media (1.4x HL-5, 8% FM) plus penicillin (60 U/ml), streptomycin sulfate (60 µg/ml), and plus or minus G418 (15 µg/ml), depending on whether the cells are transformed with an episomal plasmid. Ax2-214 and JH10 derivatives with episomal plasmids were grown in 10 µg/ml G418 or 4 µg/ml blasticidin, as appropriate. DH1, 24EH6, and p69C5-3G1 derivatives with episomal plasmids were grown in 7.5 µg/ml G418. Cells were propagated at 22°C on 10-cm plates. For suspension growth assays, cells were grown in 25-ml culture volumes in 250-ml Erlenmeyer flasks at 180 rpm. Cell densities were determined using a hemacytometer.

Mutagenesis and Mutant Screening of D. discoideum Cells
For mutagenesis, we modified the protocol described by Patterson and Spudich 1995. Log phase ORF+7-3 cells were harvested and resuspended in MES starvation buffer (50 mM MES, pH 6.8, 2 mM MgCl₂, 0.2 mM CaCl₂). 4-nitroquinolone-N-oxide (NQNO; Sigma-Aldrich) was added to 12 µg/ml. Cells were incubated in the NQNO for 30 min with gentle rocking. 35% of the cells survived at this dose of NQNO. Cells were washed twice in 5 ml of standard HL-5 media. Cells were plated at a density of 5,000 cells/well in 26 6-well tissue culture plates (Falcon; Becton Dickinson). Approximately 273,000 genomes were examined. Cells were grown for 3 to 4 d at 22°C and then shifted to 13°C for 3 to 4 d. Using a 32x magnification, wells were screened for clusters of cells with an increased number of large cells. The clusters were transferred to a fresh 6-well plate and grown up as before. The cells were passaged and screened 3 to 4 times until they appeared to be clonal. The cells were then plated on Klebsiella aerogenes lawns to select for single clones and to assay for developmental phenotypes.

For further examination of a cytokinesis phenotype, cell lines were grown in suspension culture in 25-ml cultures in 250-ml Erlenmeyer flasks at 180 rpm. We find that growth under these conditions increases the penetrance of any cytokinesis defect, resulting in multinucleated cells. Cell lines were analyzed for the nuclei/cell ratio distribution, growth rate, smoothness of the growth curve, and saturation density. A high nuclei/cell ratio and a lower saturation density are indicative of a cytokinesis defect.

Plasmids
The expression vector used for library expression, pLD1A15SN, was modified from a vector, pLD1, which contains the Ddp2-based origin of replication (Chang et al. 1990; Slade et al. 1990). The vector contains the actin-15 promoter with no ATG and the actin-15 transcription termination region. The following polylinker was introduced:

A GFP (S65T)-tagging cassette was generated in pBlueScript-II+SK that allows one to easily construct cDNA fragments with the GFP sequence fused at the 5' or 3' end for NH₂-terminal or COOH-terminal GFP fusions, respectively. The GFP (S65T)-tagging cassette can be moved easily as a SalI-NotI fragment into pLD1A15SN. In this manner, the GFP fusion proteins described below were constructed.

pLD1A15SN:GFP-dynacortin and pLD1A15SN:coronin-GFP were constructed using PCR, cloning into the GFP (S65T)-tagging cassette and subcloning into pLD1A15SN. A blasticidin resistant-dynacortin 2B19 expressing plasmid was generated using PCR to generate a cDNA with appropriate restriction consensus sites and subcloning the fragment into a modified pDXA-3H vector (Manstein et al. 1995) called pDXA-3H-Bl. This vector has had the G418-resistance marker replaced with a blasticidin-resistance marker (a gift from G. Cavet and J.A. Spudich).

Library Construction
A D. discoideum expression library was generated using the pLD1A15SN expression vector (Fig. 1). Total RNA was purified from axenically surface-grown and suspension-grown Ax2 cells, using the TRIzol extraction method (GIBCO BRL). The RNA from the two populations was quantitated by ultraviolet optical density spectroscopy and mixed in a 50:50 ratio. PolyA+ RNA was purified by selecting two times sequentially with oligo(dT)-cellulose type 2 (Collaborative Research Inc.) using standard techniques. The RNA quality was verified by appearance on an ethidium bromide-stained formaldehyde-agarose gel and by hybridization with a single labeled 3-kb gene to the RNA immobilized on a nylon membrane.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Map of the D. discoideum expression cDNA library. Underlined restriction sites are unique unless present in a given cDNA.

Library plasmid DNA was generated from 700,000–800,000 colony forming units. Colonies were allowed to grow on the plates and were harvested. Plasmid DNA was recovered, divided into two parts, and purified on a Maxi-Prep ion exchange resin (Qiagen Inc.).

Transformation of the Plasmid Library into D. discoideum Cells
In a typical experiment, mutant D. discoideum cells were grown on 20 10-cm plates in Hans' enriched HL-5 and collected during log phase growth. Cells were pooled and washed one time in 10 ml ice-cold, standard electroporation buffer (10 mM potassium phosphate, pH 6.5, 50 mM sucrose). Cells were quantitated using a hemacytometer and were resuspended at a density of 10⁷ cells/350 µl of standard electroporation buffer. 1 µg of plasmid cDNA library was transformed per 350 µl of cells. Transformation was performed by electroporation using a Gene Pulser (BioRad). Cells were pulsed one time in a 0.4-cm cuvette at 3 µF. The optimal field strength was determined for each cell line (2,500 to 3,250 eV/cm). After electroporation, 1 ml of ice-cold HL-5 was added to allow the cells to recover. Cells were grown for 20–24 h at 22°C in Hans' enriched HL-5, then the media was replaced with Hans' enriched HL-5 with the appropriate concentration of G418. The media was changed every 2–3 d until the clones were harvested. For the ORF+ derived strains, a lower limit of 1,500 colonies/µg of DNA was typically recovered. For this estimation, only the largest colonies were counted to prevent overestimation due to counting small, sibling satellite clones. This means that the actual number of colonies recovered per microgram of DNA might have been much greater. This conservative estimate was used for estimating the coverage of the cDNA library.

Selection of Suppressors
After the D. discoideum library clones reached near confluency on 10-cm plates, the cells were harvested and combined into eight distinct pools of 30,000–35,000 clones per pool. Therefore, a total of 240,000–280,000 independent clones were analyzed in the selection experiment. One seventh of each pool was subjected to growth selection. The aliquots of cells used for selection were grown in 100-ml cultures in 1-liter Erlenmeyer flasks at 180 rpm. The cells were grown and split every 3 to 4 d initially, and then every 6 to 7 d. Initial splits were 1 to 2, and were then 1 to 4, 1 to 6, 1 to 10, 1 to 30, 1 to 50, 1 to 100, 1 to 500, and finally 1 to 1,000. Growth rates of the cells were monitored throughout the entire selection. The pools were selected for 1.5–2.5 mo. This amount of time was required for the winners to emerge from the population since the mutant 11-5.1 cells can grow slowly in suspension culture, and since the winners had to emerge from a background of 30,000–35,000 clones. Once the cultures were being split at 1 to 1,000 every 6 to 7 d and the cell morphology appeared more wild-type, DNA was harvested using the Wizard preparation protocol (Promega). DNA was transformed into STBL2 cells (GIBCO BRL) and 18 clones were selected for alkaline lysis DNA preparation and restriction endonuclease digestion. Appropriate clones were selected and the sequence of the DNA was determined using standard procedures. Recovered clones were transformed into the original mutant cell line to verify that the DNA construct could recapitulate the suppression effect.

Growth Rate and Nuclei/Cell Ratio Determination
Relative growth rates were determined by diluting the cells to a similar starting density and measuring the cell densities over several subsequent time points with a hemacytometer. The cell densities were plotted versus time and the resulting log phase curve was fit to a single exponential equation using KaleidaGraph (Synergy Software). During the log phase of growth, aliquots of cells were removed, fixed with –10°C methanol, and washed in 1x PBS, 0.1% Triton X-100 (PBT). Alternatively, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in HL-5 media for 10 min, and then for 50 min in 4% paraformaldehyde in 1x PBS, followed by 3 min in –10°C acetone. Cells were washed in 1x PBS. Cells were stained with DAPI (1 µg/ml; Molecular Probes) for 20 min with gentle rocking. After washing, the cells were examined by fluorescence microscopy using a microscope (Axiovert; ZEISS). To determine the nuclei/cell ratios for strains grown on surfaces, cells were plated at low density (2–5 x 10⁵ cells/ml) and grown for 2 d. They were then fixed using the paraformaldehyde procedure described above. The number of nuclei per cell was recorded for a few hundred cells.

Genomic DNA Analysis
Genomic DNA was prepared from wild-type (ORF+7-3) and 11-5.1 cells. Genomic DNA was digested singly with EcoRI, HindIII, and XbaI, and doubly with HindIII/XbaI and EcoRI/XbaI restriction endonucleases. The DNAs were separated on a 1% agarose gel and depurinated, denatured, and neutralized before transferring to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) by capillary transfer using standard protocols. Immobilized DNAs were hybridized with denatured -³²P–labeled DNA probes using the random hexamer extension protocol (Feinberg and Vogelstein 1983). Hybridized fragments were detected using a PhosphoImager (Molecular Dynamics) and autoradiography. For cortexillin I, the 840-bp SalI/NotI cDNA insert from cortI 2A19 was used as a probe. For dynacortin, the SalI/NotI cDNA from a full-length dynacortin cDNA clone was used.

RNA Analysis
Total RNA was isolated using the TRIzol method (GIBCO BRL). RNA was quantitated using a DU 640 spectrophotometer (Beckman Coulter), separated on a 1% agarose-formaldehyde gel, and transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) by capillary transfer. DNA probes were labeled as above. Immobilized RNAs were hybridized with denatured -³²P–labeled DNA probes in Church's buffer (250 mM sodium phosphate, pH 7.0, 7% SDS, 1% BSA, 1 mM EDTA). Hybridized RNAs were detected by autoradiography.

Isolation of Full-length cDNAs and Sequence Analysis
Full-length cDNAs were isolated from the cDNA library described above. Colonies were plated on Luria broth agar plates, transferred to Hybond-N+ nylon membrane, and hybridized with radiolabeled probes. Plasmid DNA was isolated from the cells and the sequence of the insert was determined. Because the vector backbone is a D. discoideum expression vector, the full-length cDNA plasmids could be reintroduced into D. discoideum directly to test the in vivo function of the full-length cDNA. DNA and protein sequence analysis was performed using SeqWeb (Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI), and the Expasy Proteomics Tools web site (www.expasy.ch). BLAST searches (Altschul et al. 1997) were also conducted either through the SeqWeb interface or through the NCBI web site (www.ncbi.nlm.nih.gov).

Antibody Production and Western Analysis
The entire dynacortin 2B19 coding sequence (encoding amino acids 118–354) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion using the pGex-1 vector. The GST:2B19 protein was purified on glutathione resin (Sigma-Aldrich), using standard conditions. Rabbit polyclonal antibodies were generated using a standard protocol for antibody production.

For Western analysis, cell lysates were prepared by boiling cells in SDS sample buffer. Proteins were electrophoretically separated on SDS-polyacrylamide gels and transferred to nitrocellulose. The dynacortin protein was detected using the antidynacortin rabbit polyclonal antibody and a goat anti–rabbit, HRP-conjugated, secondary antibody (BioRad). The coronin protein was detected using the anticoronin mAb (deHostas et al. 1991) and a goat anti–mouse, HRP-conjugated, secondary antibody (BioRad). Immune complexes were detected using the ECL detection system (Amersham Pharmacia Biotech).

Cell Fractionation
Log phase cells were recovered and washed in 10 mM Tris, pH 7.4. Cells were resuspended in lysis buffer (75 mM NaCl, 150 mM Hepes, pH 7.1, 3 mM EDTA, 2 mM EGTA, 0.1 mM PMSF, 55 µg/ml TLCK, 80 µg/ml TPCK, 2 µg/ml leupeptin, 5 µg/ml pepstatin) ± 20 mM sodium vanadate (Na₂HVO₄; referred to as NaV_i). Cells were lysed by 3 cycles of freezing in liquid N₂ and immediately thawing. Cells were centrifuged in a TLA 120.1 rotor (Beckman Coulter) at 100,000 g for 30 min. Supernatant and pellet fractions were analyzed by SDS-PAGE and Western analysis as described above.

For complex sizing analysis, the 100,000-g supernatants were prepared in a modified lysis buffer (20 mM NaCl, 150 mM Hepes, pH 7.1, 2 mM EDTA, 2 mM EGTA, 20 mM NaV_i, and the same protease inhibitor cocktail). Samples were applied to a Superdex S200 HR 10/30 column (Amersham Pharmacia Biotech) and chromatographed using an MPLC system (Waters Corp.). The running buffer used for the gel filtration column was the same buffer without the 20 mM NaV_i and the protease inhibitors.

Immunocytochemistry
For in situ localization of the endogenous dynacortin, wild-type ORF+7-3, or Ax2 D. discoideum cells were plated on coverslips and allowed to adhere for 4 h to overnight. Cells were either fixed in –10°C methanol for 3 min or in 4% paraformaldehyde (Electron Microscopy Sciences) in HL-5 for 10 min followed by 50 min in 4% paraformaldehyde in 1x PBS, then –10°C acetone for 3 min. Cells were blocked for 10 min in 1x PBS, 0.5% BSA (PBT). If cells were fixed in –10°C methanol, 0.05% Triton X-100 was added to the PBT. Cells were immunostained with the antidynacortin polyclonal antibodies (1:1,500) in PBT. Antibodies were detected by indirect immunofluorescence using TRITC or FITC-conjugated donkey anti–rabbit secondary antibodies (Molecular Probes) as appropriate. For nuclei staining, 4',6'-diamidino-2-phenylindole (DAPI; Molecular Probes) was added at a concentration of 1 µg/ml and for filamentous actin staining, rhodamine-phalloidin (Sigma-Aldrich) was added at 0.1 µg/ml. All washes were also done in either 1x PBS or PBT.

Microscopy
Cell imaging was done with a 40x (NA 0.75) or a 63x objective (NA 1.3) on a microscope (Axiovert; ZEISS). Images were collected using the Metamorph Software package (Universal Imaging Corp.) and images were processed with Adobe Photoshop (Adobe Systems Inc.). Ratio imaging was performed by determining the ratio of the maximal pixel intensity of the protrusion (Max. I [protrusion]) and the maximal pixel intensity of the lateral membrane (Max. I [lateral membrane]). To determine the number of protrusions of the cell, we examined the number of protrusions present on the cell at 30-s intervals. This provides a dynamic factor and allows periodic quiescent cells to be represented. The counts were determined from a single optical section of the cells so that protrusions on the extreme dorsal surface may be missed.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A Genetic Screen Generated Cytokinesis-deficient Strains
We conducted a genetic screen for cytokinesis-deficient cell lines by chemically mutagenizing cells with 4-nitroquinolone-N-oxide and screening plates of cells for clusters of enlarged, multinucleated cells. These clusters of cells were isolated and clones were purified. The strains were subjected to growth in suspension culture, a powerfully selective condition against cytokinesis-deficient growth. The clones were also tested for the ability to complete normal development. One cell line referred to as 11-5.1 had a strong cytokinesis defect when grown in suspension culture (Fig. 2). This strain had an increased propensity to form multinucleated cells on surfaces and formed small, abnormal sorocarps (fruiting bodies) on Klebsiella aerogenes lawns. We used this strain in a library complementation and multicopy suppression experiment.

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Phenotypic characterization of a cytokinesis-deficient mutant. A, A phase microscopy image of fixed, parental wild-type cells grown in suspension culture shows that these cells are small and uniform in size. B, DAPI staining of these wild-type cells in A reveal that the cells are largely mononucleated. C, Wild-type cells grown on surfaces are mostly small and uniform. D, Wild-type cells form large sorocarps when grown on K. aerogenes lawns. E, A phase microscopy image of a fixed, 11-5.1 cell grown in suspension culture shows that these cells are greatly enlarged. F, DAPI staining of the cell in E shows that it is highly multinucleated. G, 11-5.1 cells grown on surfaces have a higher proportion of enlarged cells. Long connections between the large cells and a daughter cell that is forming by traction-mediated cytofission is often observed. H, 11-5.1 cells, when plated on K. aerogenes lawns, form sorocarps that are reduced in size. Bars: (A–C, E–G) 10 µm; (D and H) 500 µm. Wild-type for these experiments was the ORF+7-3 strain.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Characterization of rescue of 11-5.1's growth in suspension by cortI 2A19, 2B19, and 4C2. A, Introduction of these plasmids into fresh 11-5.1 cells allows rescue of the growth rates and saturation densities of these cells compared with 11-5.1 transformed with pLD1A15SN with no insert. The wild-type and 11-5.1 control cells carried the parental vector, pLD1A15SN, with no insert. A compilation of growth experimental data is shown in Table . B, Analysis of the distributions of the number of nuclei/cell for each strain shows that cortI 2A19, 2B19, and 4C2 dramatically improves the distribution of nuclei/cell, increasing the number of mononucleated cells. Number of cells counted for each genotype was as follows: wild-type:pLD1A15SN, 175; 11-5.1:pLD1A15SN, 271; 11-5.1:cortI 2A19, 215; 11-5.1:2B19, 197; 11-5.1:4C2, 272. In other growth experiments, the cellular morphology, which is an effective indicator of the nuclei/cell distribution, was clearly rescued as determined by viewing the cells in the microscope. C, For comparison, the nuclei/cell distributions of cells grown in suspension from the rescued pools Tf2A, Tf2B, and Tf4C cells are shown. Data acquired from 11-5.1 cells transformed with pLD1A15SN and from pools Tf2A and Tf4C before the selection process were pooled to calculate SEM (bars) for the unrescued 11-5.1 mutant strain grown in suspension culture (11-5.1:pLD1A15SN in the histogram). The sizes of the data sets were n = 271, 132, and 124 cells, respectively. 11-5.1:Tf2A, 11-5.1:Tf2B, and 11-5.1:Tf4C were pools Tf2A, Tf2B, and Tf4C after the selection process. The data sets were based on n = 60, 92, and 108 cells, respectively. D, 2B19 recognized a 1.2-kb transcript that is present in both wild-type and 11-5.1 cells. The expected 1.4-kb cortexillin I transcript was found in wild-type, but was undetectable in 11-5.1 cells. This experiment is representative of three independent experiments using three independent preparations of RNA. E, Analysis of restriction endonuclease-digested wild-type and 11-5.1 genomic DNA revealed that most or all of the cortexillin I locus is absent in 11-5.1 cells. The immobilized, electrophoretically separated DNAs were hybridized first with a cortexillin I cDNA probe and were then reprobed with a dynacortin cDNA probe. The fragments detected with the cortexillin I probe are still visible after reprobing with the dynacortin cDNA probe. a, EcoRI digest; b, HindIII digest; c, XbaI digest, d, HindIII/XbaI double digest; e, EcoRI/XbaI double digest. 8–9 µg of genomic DNA per lane. F, Analysis of the nuclei/cell distribution of cortI cells transformed with pLD1A15SN, cortI 2A19, or 2B19 revealed that cortexillin I and dynacortin 2B19 suppressed this mutant. These cells were grown in standard HL-5 media. Presented are the mean and SEM from three to four data sets per line, where each data set typically included 100–300 cells. Wild-type for experiments in A–E was the parental ORF+7-3 strain. Wild-type for the experiment in F was the parental line, Ax2-214.

To verify that the reversion in the rescued lines is due to the plasmid isolated from the cells, the plasmids, cortI 2A19, cortI 2A5, 2B19, and 4C2, were transformed into fresh 11-5.1 cells (Fig. 3A and Fig. B; Table ). Both cortI 2A19 and cortI 2A5 rescued 11-5.1's growth in suspension, including growth rate, saturation density, and cell morphology (Fig. 3A and Fig. B; Table ). 2B19 and 4C2 also rescued 11-5.1's growth in suspension according to all of the criteria used (Fig. 3A and Fig. B; Table ). Rescue by all of these constructs was apparent within a few days after being placed under suspension growth conditions and they rescued immediately after introduction into fresh 11-5.1 cells. In other words, long periods of selection were not required for rescue by the plasmids upon reintroduction into 11-5.1. The coronin antisense construct, coronAS 24, when introduced by itself, did not rescue 11-5.1, but instead induced a strong coronin^– phenotype (deHostas et al. 1993) in wild-type, 11-5.1, and myo II heavy chain (myo II^–) mutant cells (Table ).

Intriguingly, the list of plasmids described above were the only plasmids recovered from rescued pools with one exception. One clone of discoidin Ic (Devine et al. 1981) was recovered from a fourth pool, Tf4D, using the developmental assay. Reintroduction of the plasmid was able to rescue the development of the 11-5.1 cells when plated at high density, but not low density (plated for single discrete plaques). It did not, however, rescue growth in suspension in the original transformant or when reintroduced into fresh 11-5.1 cells. It was not studied further.

Examination of mRNAs and Genomic DNA Indicates 11-5.1 Is an Allele of Cortexillin I
To determine which of the genes was mutated in 11-5.1, we began by examining the mRNA levels of each of the genes in 11-5.1 cells compared with the parental strain (Fig. 3 D). Since the cortexillin I constructs and the 2B19/4C2 constructs rescued 11-5.1's growth in suspension to wild-type levels, we began examining these two genes first. The 1.4-kb cortexillin I mRNA was completely absent in 11-5.1 cells, but present in the parental wild-type cells. The 1.2-kb 2B19/4C2 mRNA was present in both strains. In addition, hybridization of restriction-digested genomic DNA with a cortexillin I cDNA probe revealed that the majority of the cortexillin I locus is absent in the 11-5.1 strain, whereas the dynacortin locus appeared to be normal in both strains (Fig. 3 E). Since we were able to rescue all of the defects (growth in suspension and development) of 11-5.1 with cortexillin I constructs and all or most of the cortexillin I genomic locus and its transcript are absent in 11-5.1, 11-5.1 is an allele of cortexillin I. Strain 11-5.1 will be referred to as cortI^11-5.1.

Dynacortin 2B19 Suppresses an Independent Allele of cortexillin I
To test whether cortI 2A19 and dynacortin 2B19 could complement an independent cortexillin I mutant cell line, both constructs were transformed into an Ax2-derived strain in which the cortexillin I locus had been deleted by homologous recombination (cortI; Faix et al. 1996). Both plasmids rescued cortI cells; however, the 2B19 plasmid offered a modest, but significant, rescue of the growth properties of this strain when the cells were grown in Hans' enriched HL-5 media (Table ). Both plasmids rescued to a higher level when the cells were grown in standard HL-5 media (Fig. 3 F, Table ). The differences in the growth properties of the cortI cells under the two media conditions suggest that these cells are more sensitive to environmental conditions; it will be useful to determine whether that is a general property of cytokinesis-impaired strains. Furthermore, since dynacortin 2B19 suppressed this strain, it indicates that its suppression of the loss of cortexillin I is general and not specific to a particular genetic background.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Sequence features of dynacortin. A, The dynacortin gene encodes a protein that has high serine and threonine content. There are also multiple consensus phosphorylation sites. Consensus casein kinase II sites are in bold, consensus protein kinase C sites are underlined, and the only consensus protein kinase A site is italicized. Box 1 encloses a candidate PEST sequence, which has a PEST search score of 12.34 (Rogers et al. 1986). Box 2 encloses an -helical, hydrophobic patch at the COOH terminus. B, A cartoon of the linear relationship between the PEST sequence, the COOH-terminal hydrophobic patch, and the phosphorylation sites (asterisks) is presented. The region encoded by dynacortin 2B19 (amino acids 173–354) is shown as a gray box. The dynacortin sequence is available (GenBank/EMBL/DDBJ accession number AF222688).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Dynacortin is a soluble protein that forms a high molecular weight complex. A, Dynacortin is a soluble protein that might contain phosphates. Western analysis of cell lysates prepared by boiling in SDS-PAGE sample buffer (WCL) reveals a protein with an apparent molecular weight of 48 kD. Dynacortin partitions into the 100,000-g supernatant, but has a faster mobility more similar to the expected 38-kD mol wt calculated from the primary sequence when the cell lysates are prepared in the absence of sodium vanadate (–NaVi lanes). The addition of 20 mM sodium vanadate (+NaVi), an inhibitor of phosphatases, blocks this mobility shift, suggesting that the mobility shift is due to dephosphorylation of the protein upon cell lysis. The dynacortin species in the WCL lane and the dynacortin species in the +NaVi supernatant lane comigrate when mixed, verifying that these are the same molecular weight forms (not shown). S, 100,000-g supernatant; P, 100,000-g pellet. B, The wild-type soluble dynacortin complex migrates on a gel filtration column comparable to a globular 250–300-kD complex. The supernatant analyzed in B was prepared from native 100,000-g supernatants from wild-type ORF+7-3 cells carrying the pLD1A15SN plasmid with no insert. C, The wild-type complex is disrupted by the expression of the dynacortin 2B19 version. The complex molecular weight is 100 kD smaller, and the 2B19 version is 50 kD, larger than expected for the 20-kD monomer, suggesting that it may be titrating out a cofactor. The supernatant analyzed in C was prepared from native 100,000-g supernatants from ORF+7-3 cells expressing dynacortin 2B19. Retention time in B and C is in minutes.

We also tested for dynacortin complex formation in cortI^11-5.1 cells and RacE cells and in both cases the protein complex had the wild-type retention time (data not shown). In other cell fractionation experiments, we observed that dynacortin and coronin fractionated separately even though the core dynacortin complex remained assembled (data not shown). Therefore, RacE, cortexillin I, and coronin are not likely to be present in the dynacortin core complex.

Overexpression of Dynacortin Induces a Cytokinesis Defect
We introduced the full-length dynacortin gene into wild-type cells, cortI^11-5.1 cells, and myo II^– cells. In all of the strains, expression of full-length dynacortin induced a prominent cytokinesis defect when the cells were grown on surfaces (Fig. 6). Interestingly, the cells sensitive to overexpression of dynacortin could overcome this defect after several weeks in culture, even though they still overexpressed the protein (data not shown). 2B19, on the other hand, did not induce a dominant defect in cytokinesis. The full-length version of dynacortin failed to rescue the growth in suspension of cortI^11-5.1 cells even after growing the cells in suspension for a few weeks (a period of time much longer than required for cortI 2A19 and dynacortin 2B19 to rescue cortI^11-5.1) to select for rescued subclones. This indicates that suppression of cortI^11-5.1 is specific to the dynacortin 2B19 fragment (data not shown). This fact, combined with the result that dynacortin 2B19 disrupted the endogenous dynacortin complex, suggests that dynacortin 2B19 might antagonize the wild-type dynacortin pathway to suppress the loss of cortexillin I. We verified the expression of full-length dynacortin and the 2B19 domains by Western analysis on total cell protein lysates from each cell line (Fig. 6 A). Full-length dynacortin was overexpressed relative to the endogenous protein in all of the strains examined. The dynacortin 2B19 construct also produced a protein that was overexpressed relative to the endogenous dynacortin.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Dynacortin induces a dominant cytokinesis defect when overexpressed. A, Western analysis of full-length dynacortin and dynacortin 2B19 proteins in the different cell lines reveals that appropriate sized proteins are indeed expressed and that full-length dynacortin is overexpressed in each of the cell lines. Dynacortin 2B19 is easily detected in myo II– cells with slightly longer exposure times (not shown). B, Expression of dynacortin 2B19 in wild-type, cortI11-5.1, and myo II– cells does not induce any deleterious effect. Overexpression of the full-length version of dynacortin in wild-type, cortI11-5.1, and myo II– cells induces a dominant cytokinesis defect as indicated by the large, multinucleated cells when grown on surfaces. Bar, 10 µm (applies to all panels). C, The overexpression defect was quantitated by determining the distribution of cells containing each number of nuclei per cell revealed by DAPI staining. Since the growth on surfaces is a more permissive growth condition, the defect caused by the full-length dynacortin is severe. Overexpression of dynacortin in these strains caused this defect in all (three or more independent transformations for each strain) experiments. The following number of cells were counted for each genotype for the data set presented: wild-type:pLD1A15SN, 288; wild-type:dyna 2B19, 300; wild-type:dynacortin, 374; cortI11-5.1:pLD1A15SN, 289; cortI11-5.1:dyna 2B19, 234; cortI11-5.1: dynacortin, 192. Wild-type for these experiments was the ORF+7-3 strain.

Dynacortin Is Enriched in Dynamic Domains of the Cell Cortex
We used the dynacortin polyclonal antibodies to identify the cellular localization of endogenous dynacortin by immunocytochemistry (Fig. 7A and Fig. B). Dynacortin localized to the cortex and was found to be enriched at dynamic domains of the cortex, including pseudopodia (shown in Fig. 7 A), filopodia, lamellipodia, and macropinocytotic crowns. A population of dynacortin can also be detected in the cytoplasm. Hence, we named the protein dynacortin for its enrichment at dynamic cortical domains. Regions of the cell cortex that were enriched for dynacortin were also enriched for filamentous actin as expected (Fig. 7 B). We also examined dynacortin localization by expressing a GFP-dynacortin fusion protein. In addition to an apparent cytoplasmic component, GFP-dynacortin localized predominantly to the cell cortex and was enriched in protrusions in wild-type and in cortI^11-5.1 cells (Fig. 7 C). Immunodetection of dynacortin in wild-type cells expressing dynacortin 2B19 revealed a comparable dynacortin distribution, except more abundant due to the extra protein, suggesting that the dynacortin 2B19 protein localizes similarly to the endogenous protein (data not shown).

View larger version (78K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Localization of dynacortin. A, An immunofluorescence micrograph and a phase image of a wild-type D. discoideum cell reveals that endogenous dynacortin is found in the cytoplasm, is localized to the cell cortex, and is enriched at dynamic domains such as pseudopodia. This cell was fixed using the methanol procedure. B, Actin and dynacortin are coenriched in regions of cortical protrusive activity. These cells were fixed using the paraformaldehyde/acetone procedure. It should be noted that the optimal fixation conditions for preserving dynacortin are incompatible with the optimal fixation conditions for actin. The cells in Fig. 7 B were fixed under conditions that best preserve both proteins simultaneously. C, GFP-dynacortin localizes similarly to the endogenous dynacortin detected by immunocytochemistry. In both wild-type and cortI11-5.1 cells, dynacortin is found at the cortex, but is enriched in protrusions. D, GFP-dynacortin is found in the cytoplasm and is evenly distributed around the cell cortex during early cytokinesis. It becomes enriched in the polar ruffles during late cytokinesis. After separation of the two daughter cells, it immediately becomes enriched in cell protrusions (4:00). Cells in C and D are imaged alive and are expressing GFP-dynacortin. Time is in minutes:seconds. Bar, 10 µm (applies to A–D). Wild-type cells in A and C are the ORF+7-3 cell line, and the cells in B are the Ax2-214 cell line. The wild-type cell in D is derived from the DH1 strain.

Coronin Is Not Required for Dynacortin Localization
Because we recovered a coronin antisense construct in one of the suppressed strains and because coronin and dynacortin are distributed similarly in the cell cortex, we examined the relationship between coronin and dynacortin. Coronin protein expression was disrupted in wild-type cells expressing the coronin antisense construct (Fig. 8 A). This produced a coronin^– phenotype in these cells (Table II; deHostas et al. 1993). Interestingly, dynacortin, actin, and coronin all had identical cortical distributions (Fig. 8 B). Immunocytochemistry on cells expressing the coronin antisense transcript revealed that dynacortin and filamentous actin localized to the cell cortex and to cortical protrusions, indicating that coronin is not required for the localization of dynacortin to the actin-rich cortex (Fig. 8 C).

View larger version (77K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Coronin antisense and coronin dependence. A, The coronin antisense construct completely inhibits accumulation of coronin protein as determined by SDS-PAGE and Western immunodetection using anticoronin antibodies. To verify loading, protein extracts were quantitated using the BioRad protein assay and equivalent aliquots from the same samples were examined in parallel by Coomassie staining of SDS-PAGE gels and by immunodetection with antidynacortin antibodies (not shown). A similar analysis was repeated on separate protein extract preparations. B, Coronin-GFP localized to protrusive domains of the cell cortex that were also enriched for actin and dynacortin. C, Dynacortin and actin localized to the cell cortex of coronin antisense expressing cells. Coronin-GFP or coronin antisense expressing cells were fixed and stained with rhodamine-labeled phalloidin to identify filamentous actin or with antidynacortin antibodies to reveal dynacortin. Bars, 10 µm (each applies to all panels of its respective group). Wild-type for these experiments was the ORF+ 7-3 strain. These cells in B and C were fixed using the paraformaldehyde/acetone procedure.

View larger version (83K): [in this window] [in a new window] [Download PPT slide]   Figure 9 RacE is required for dynacortin and coronin localization. A, GFP-dynacortin localizes to the cell cortex and enriches dynamically in the protrusive domains of the wild-type (parental DH1) cells. B, In the RacE– (24EH6) cell, GFP-dynacortin concentrates in the protrusive domains of the cells. Numbers in A and B are in minutes:seconds. C, Coronin-GFP localizes to the cortex and is enriched in protrusions in wild-type cells. Coronin-GFP is more concentrated in cell surface projections in the RacE– cell line. Bars, 10 µm (applies to all panels in each group). All cells in A–C were imaged alive. D, Determining the ratio of the maximum pixel intensity of the protrusion [Max. I (protrusion)] to the maximum pixel intensity of the lateral membrane [Max. I (lateral membrane)] revealed that GFP-dynacortin is enriched 28% in the protrusions compared with the lateral membrane in the wild-type cells. In the RacE– cells, GFP-dynacortin is enriched 70% in the protrusions. By plotting the number of protrusions at each ratio, it is apparent that the populations are distinct distributions. E, The mean ± the SEM are displayed for the cell lines. The differences between each pair of populations are significant (t test: P < 0.0001). The ratios were determined for cells imaged for a period of time up to 10 min. Therefore, the distributions were determined by making the measurements for a total of protrusions formed over time by m cells. Wild-type:GFP-dynacortin: n = 128, m = 17; RacE–:GFP-dynacortin: n = 233, m = 11; wild-type:coronin-GFP: n = 57, m = 13; RacE–:coronin-GFP: n = 65, m = 7. F, The RacE– mutant cells also have a greater number of dynacortin- and coronin-rich protrusions on their surfaces compared with the wild-type strain (t test: P < 0.0001). Number of protrusions was determined by averaging the number of protrusions detected by a single optical view of the cells taken every 30 s for several minutes. This allowed us to accurately count time points in which the cell cortex was quiescent (no apparent protrusion). Therefore, averages were determined from a total of time points taken from m cells. Wild-type:GFP-dynacortin: n = 78, m = 4; RacE–:GFP-dynacortin: n = 81, m = 3; wild-type:coronin-GFP: n = 249, m = 13; RacE–:coronin-GFP: n = 151, m = 8. Qualitative observations from at least ten of other cells for each genotype were consistent with the quantitative results in D–F.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Genetics of Global Cortical Stiffness/Tension and Cell Shape Control
In D. discoideum, several genes are required for cytokinesis and to generate cortical stiffness of the cell, which contributes to the basal control of cell shape during interphase and morphogenesis. RacE (Larochelle et al. 1997; Gerald et al. 1998) and coronin (deHostas et al. 1993) are distributed globally around the cell throughout the cell cycle. Loss of either RacE or coronin results in a cytokinesis defect. The RacE mutant cells have reduced global cortical tension and cannot undergo cytokinesis when grown in the absence of adhesion (Gerald et al. 1998). The coronin mutant cells, in contrast, have defects under adhesive and nonadhesive conditions (deHostas et al. 1991; this paper). Since each of these proteins is distributed globally, it suggests that these proteins function in a global system of cortical tension and cell shape control that is essential for cytokinesis.

Dynacortin might also function in the global system for cell shape and cortical stiffness. During cytokinesis, dynacortin is distributed in the cortex globally and even appears somewhat depleted from the cleavage furrow and midbody. Intriguingly, dynacortin was isolated because a fragment of it encoding the COOH half (residues 173–354) suppressed the loss of cortexillin I. Since full-length dynacortin did not rescue the loss of cortexillin I, the COOH fragment of dynacortin might be antagonizing endogenous dynacortin and this antagonism may compensate for the impaired, spatially restricted cortexillin I pathway. The finding that the COOH 2B19 domain disrupts the formation of the wild-type dynacortin complex is consistent with the notion that it antagonizes the wild-type dynacortin function. Overexpression of the full-length dynacortin gene induces a cytokinesis defect in wild-type cells, cortI^11-5.1 cells, and myo II^– null cells. This suggests that myo II and cortexillin I are not required for this dominant effect.

The coronin antisense construct was recovered along with the Ncortexillin I (cortI 2A19) construct in the predominant clone of cells from the selection experiment, suggesting that a reduction in the level of coronin offered a selective advantage to the cortexillin I mutant cell line. Indeed, the coronin antisense construct inhibits expression of the coronin protein. Consistent with this model, overexpression of coronin in the cortI^11-5.1 mutant cells caused an increase in the number of large, multinucleated cells. However, overexpression of coronin in wild-type cells increased the growth rate of the cells (Table ). Thus, we cannot exclude the possibility that a cytokinesis-impaired strain might be more sensitive to an increase in overall growth rate. The myo II^– and RacE null cells were not sensitive to overexpression of coronin, arguing against this. However, these mutants are derived from different parental D. discoideum strains so one cannot rule out strain differences. An alternate interpretation of this result is that since the coronin antisense construct produced a coronin^– mutant phenotype, the Ncortexillin I construct can rescue a coronin^–, cortexillin I^– double mutant strain's defect for growth in suspension. In either case, this particular genetic interaction experiment is nonintuitive and is an example of one of the surprising results from interaction genetic studies.

Both coronin and dynacortin colocalize in the cortex and are coenriched in protrusive domains. The normal cortical localization of both proteins depends on RacE and in the absence of RacE, they become concentrated in the protrusions. It is probable that these are not the only proteins affected by RacE since other actin-binding proteins, such as myo I (Fukui et al. 1989), -actinin (Furukawa and Fechheimer 1994), and cofilin (Aizawa et al. 1997), have a related subcellular distribution. However, they provide useful diagnostic markers, which might shed light on why RacE mutants have reduced cortical stiffness. Loss of cortical proteins from the lateral cortex (regions between protrusions) may be expected to cause a reduction in cortical stiffness. Intriguingly, dynacortin and coronin are still recruited into protrusive domains efficiently in the RacE mutant cells. Other Rac-family small GTPases likely control some aspects of the protrusive activity. RacF1 becomes enriched in macropinosomes and phagosomes (Rivero et al. 1999), whereas RacE remains uniformly distributed around the cell cortex (Larochelle et al. 1997). In contrast, overexpression of RacC induces ectopic actin-rich protrusions (Seastone et al. 1998). Since many of the cortical cytoskeletal elements are shared between the lateral membrane and many of the actin-rich protrusive structures, different Rac-family proteins may compete for these cortical elements. Thus, it is possible that in the RacE mutants, the remaining Rac small GTPases do not have to compete with RacE so that more dynacortin and coronin are recruited into the cortical protrusions.

Global Stiffness/Tension versus Spatial Contractility
Conflicting points of view regarding the mechanism of cytokinesis in the literature include the model of polar relaxation (for example, White and Borisy 1983) and the model of equatorial stimulation (for review, Rappaport 1996). In the polar relaxation model, the cell equator maintains a constant cortical tension, whereas the daughter cell pole or body relaxes, allowing the cell to cleave. In the more widely accepted, equatorial stimulation model, the equatorial tension increases through a myo II-dependent mechanism. Both models invoke the formation of a tension differential where the peak resides at the equator or cleavage plane.

One hypothesis indicated by our genetic interaction study suggests a role for the contraction of the equatorial ring to overcome the basal level of global stiffness/tension (Fig. 10). myo II (Moores et al. 1996; Neujahr et al. 1997) and cortexillin I (Weber et al. 1999) are recruited to the contractile ring where they contribute to the contractility of the equator. Both myo II (Pasternak et al. 1989) and the cortexillins (Simson et al. 1998) have been shown to be required for cortical stiffness in interphase cells. During cytokinesis, if the equatorial system is impaired by mutating cortexillin I, this can be rescued by fixing the equatorial system by expressing cortexillin I. Alternatively, if dynacortin has a role in global cortical stiffness/tension and cell shape control and is antagonized by dynacortin 2B19 protein, reduction of dynacortin activity may make the global cortex more deformable for the residual equatorial contractile apparatus. While the data presented in this paper do not directly address the mechanical aspects of this hypothesis and certainly other hypotheses can be envisioned, this idea does help to reconcile how a mutant strain defective for contractile ring function can be suppressed by a cortical protein that is enriched in the polar cortex.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 10 A model of the circuitry of proteins required for cell cleavage. A, A preliminary genetic map for cytokinesis is suggested by multicopy suppression of a cytokinesis-deficient strain of D. discoideum. Dynacortin, coronin, and RacE might function in a global pathway for cortical stiffness/tension and cell shape control. Localization of dynacortin and coronin depends on RacE, whereas the localization of dynacortin is independent of coronin. Dynacortin and coronin are drawn on separate branches, although future experiments might uncover a link between them. It is not known how many steps exist between RacE and coronin or dynacortin. Cortexillin I and myo II function in an equatorial pathway to generate spatially restricted contractility. B, A summary of the localization of myo II, cortexillin I, RacE, dynacortin, and coronin during cytokinesis is shown. C, A hypothesis about how the genetic data in this paper might relate to cytokinesis. Cortexillin I and myo II are required at the contractile ring to generate contractility at the furrow. This must overcome a global pathway, breaking the uniformity or symmetry of the cortex for cleavage. Dynacortin may participate in this global pathway that must be overcome by the contractile ring components for cell cleavage. If the equatorial system is impaired (cortI–), the equatorial contractility is reduced so that it cannot overcome the global system. If the global system is antagonized or lowered (perhaps in cortI–:dynacortin 2B19), the residual contractile system at the equator may now overcome the global pathway, leading to more efficient cell cleavage.

	Acknowledgments

 
We thank Denis Larochelle and Arturo DeLozanne for providing us with the parental and RacE mutant cell lines and the RacE cDNA. We thank Eugenio DeHostas for anticoronin antibodies and Jan Faix for the cortexillin I deletion strain. We thank members of the Spudich lab for numerous helpful discussions and in particular, Hans Warrick for training in D. discoideum techniques.

This work was supported by the National Institutes of Health grant #GM40509 to J.A. Spudich and by a Runyon-Winchell fellowship (#DRG1457) to D.N. Robinson.

Submitted: 7 March 2000

Revised: 20 June 2000

Accepted: 28 June 2000

Abbreviations used in this paper: myo II, myosin II; myo II^–, myo II null cell line; NQNO, 4-nitroquinolone-N-oxide; NaV_i, sodium vanadate (Na₂HVO₄); ORF, open reading frame.

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