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Abstract |
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Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2+, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2 cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.
Key Words: microtubule, Schizosaccharomyces pombe, cytoskeleton, kinesin, cell polarity
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Introduction |
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A particular shape and a well-defined polarity are characteristic of many cell types, as illustrated by the extended morphology of differentiated nerve cells or the asymmetric organization of polarized epithelia. The components of the cytoskeleton play an essential role in establishing and maintaining the morphology of interphase eukaryotic cells (
The interphase microtubule network is generally dynamic, with the addition and loss of tubulin subunits occurring mostly at one microtubule end, the "plus" end (for a review see
Many proteins affect microtubule stability and length, including microtubule-associated proteins (MAPs), kinesin-like proteins (klps),1 and microtubule-severing enzymes (for a review see
The unicellular fission yeast Schizosaccharomyces pombe offers a useful model system in which to study the molecular mechanisms that control eukaryotic cellular morphology because it is amenable to detailed morphological, genetic, and molecular analyses. After cell division, growth begins only at the old end of the cell. Early in G2, growth is also initiated from the new end of the cell (
Although the actin cytoskeleton appears to be needed for the actual deposition of growth material (
We have sought cellular components that work in conjunction with the microtubule cytoskeleton to establish and maintain cellular polarity. Through the molecular identification of tea2+, a gene identified in a screen for morphology mutants and shown to be required for normal behavior of the cell's growing tip ( and tea2-1 cells often establish an ectopic growth site resulting in the formation of T-shaped cells. Likewise, long cells are particularly sensitive to the loss of tea2+. Tea2p localizes to cell tips and is also often seen as dots coincident with cytoplasmic microtubule ends.
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Materials and Methods |
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Strains and Cell Culture
All strains used are shown in Table 1. Strains were constructed and maintained as described in Moreno et al., 1991. Cultures were grown in rich medium containing yeast extract plus supplements (YES) or a Edinburgh minimal medium (EMM;
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PCR Screen for klps
Primers to conserved portions of the kinesin motor domain were used to amplify genomic DNA. Genomic DNA was prepared as described in Moreno et al., 1991. The 5' primers were TAC/TGGNCAA/GACNGG (corresponding toYGQTGSGK) or TAC/TGGNCAA/GACNGG (corresponding to YGQTGTGK), and the 3' primer was C/TTCNG/CA/TNCCNG (corresponding to DLAGSE). PCR amplifications were performed on three different samples of DNA: (a) genomic DNA from wild-type cells; (b) genomic DNA from wild-type cells digested with XbaI, which restricts within pkl1 ( cells. Reaction conditions were 30 cycles of 95°C for 30 s, 40 or 45°C for 30 s, and 72°C for 30 s, generally followed by a single 5-min incubation at 72°C. PCR products were subcloned and analyzed by colony PCR. To identify clones that represent previously identified klps, colony PCR products were digested with enzymes that cut within cut7+ (
Cloning of tea2+ by Mapping and Complementation
tea2+ was cloned by positional mapping and complementation (
To show that 14T contained tea2+ and not an extragenic suppressor, the clone was integrated into the genome by homologous recombination, and this strain was crossed to leu1-32 and tea2-1 leu1-32 strains. 14T was mapped to within 0.3 cM of tea2-1 and the rescuing activity to within 0.5 cM of LEU2, demonstrating that the tea2-1 rescuing activity is linked to 14T and that the site of integration is very close to tea2-1.
Molecular Characterization of the tea2+ Region
Unless otherwise specified, molecular biology techniques are essentially as described in Sambrook et al., 1989, and sequencing was performed at the University of Colorado automated DNA sequencing facility. A genomic library, provided by A. Carr (
To isolate DNA further 3' of the tea2+ ORF, the 11-kb XhoI fragment extending 3' from the tea2+ ORF was cloned by constructing and screening an XhoI genomic library of 11-kb XhoI genomic fragments cloned into pBluescript. This cloned region, cloneX/X, was digested with XhoI and BamHI, and the 4.3-kb XhoI-BamHI fragment (see Fig 1 A) was cloned into the XhoI/BamHI sites of pBluescript. This clone, X/B, was used for sequencing and for Northern blot analysis.
A 1.4-kb AvaI/ HindIII fragment containing the 3' half of tea2+ ORF was used to screen a cDNA library (provided by F. LaCroute, Centre de Genetique Moleculare Gif sur Yvette, France). Approximately 60,000 clones were screened, and one cDNA was identified. The cDNA was excised using NotI and were inserted into the NotI site of pSPORT to construct pSPORTtea2+ cDNA, and this clone was sequenced.
Total RNA was isolated from wild-type S. pombe cells as described in Moreno et al., 1991. Poly(A+) RNA was isolated using GIBCO BRL oligo(dT) cellulose columns according to the manufacturer's recommendations. Northern blot analyses were performed as described in
Reverse transcription followed by PCR (RT-PCR) was performed using the Promega Access RT-PCR kit. Total RNA was used as template with the primer 5'-CGTAGTATATGATTGTAGCAGGTCGTC-3' for reverse transcription and the primer combination 5'-CGTAGTATATGATTGTAGCAGGTCGTC-3' and 5'-CTGTGACTCAGGAAACGCAACTTC-3' for PCR.
Computer-aided Sequence Analysis
The BLAST program available at http://www.ncbi.nlm.nih.gov/BLAST/ was used for sequence searches. The BestFit program from the GCG sequence analysis package was used for direct sequence comparison. For phylogenetic analysis, the 340amino acid (aa) motor domains of Tea2p and 42 other klps were aligned using the ClustalW program (
Construction of Knockout Strain
A null allele of tea2+ was constructed by single-step gene replacement protocol, replacing the tea2+ ORF with his3+ by homologous recombination. To construct the integration plasmid, clone 1124 was digested with BbsI, blunt ended with Klenow, then digested with HindIII, and the 423-bp [BbsI]-HindIII fragment beginning 14 bp 3' of tea2+ ORF was isolated. A SalI/SmaI fragment containing his3+ was isolated from pAFI (
Identification of tea2+ and klp4+ as the Same Gene
The gene initially characterized as klp4+ was found to be entirely contained within the 14T plasmid using PCR primers specific to klp4+. Clone 14T was used to construct 5' and 3' deletions to further define the rescuing region (see Fig 1 A). 14B and 14H are 3' truncations with deletions extending to the BamHI and HindIII sites, respectively. 14X is a deletion with 5' sequences removed to the XhoI site. The tea2-1 allele was sequenced by PCR amplification of tea2-1 genomic DNA using primers specific to the tea2+ region followed by sequencing of the PCR products.
Inducing Cells to Exit from Stationary Phase
Cells were grown in YES or EMM at 32°C until they reached stationary phase growth, generally 1 d beyond logarithmic growth in YES or 2 d after logarithmic growth in EMM. Cells were then diluted 1:10 or 1:25 in fresh medium and examined by microscopy at various times after dilution. For tea2 complementation tests, cells were grown to saturation in EMM with appropriate supplements. For lineage analysis, cells were grown to saturation in YES, placed on a YES agar pad (YES medium with 2% agar) on a microscope slide, and examined by differential interference contrast (DIC) microscopy using a Zeiss microscope. The slide was warmed to 32°C with an air curtain incubator (Sage Instruments) or a heatlamp. The temperature was controlled using a CN76000 microprocessor-based temperature and process controller from Omega Engineering. Images were captured using an Empix charge-coupled device camera and Metamorph software (Universal Imaging).
Production of Antibodies
For protein expression in Escherichia coli, a construct was made by digestion of clone 424 with BsmI and BbsI and generation of blunt ends with T4 DNA polymerase and Klenow. The 440-bp [BsmI-BbsI] fragment corresponding to the COOH-terminal region of Tea2p (which lacks motor sequences) was cloned into the EcoRI site of pGEXKG (
A second fusion protein was constructed for antibody purification. The 440-bp [BsmI-BbsI] tea2+ fragment described above was cloned into the PvuII site of pRSETc (Invitrogen) and the fusion protein expressed in the BL21(DE3) E. coli strain. The fusion protein was solubilized by denaturization and then purified by chromatography on nickel columns according to the procedure recommended by Invitrogen. A column for affinity purification was made with purified fusion protein covalently cross-linked to cyanogen bromideactivated sepharose 4B (Sigma-Aldrich) as recommended by Amersham Pharmacia Biotech. The serum was purified on the column essentially as described in
Immunofluorescence Microscopy
Cells were prepared for immunofluorescence staining by aldehyde or cold methanol fixation as described in Hagan and Hyams, 1988. For tubulin staining, a mouse moncolonal antibody against Drosophila -tubulin was used (provided by M.T. Fuller, Stanford University, Stanford, CA) or tat1 (
Immunofluorescence microscopy was performed on a Leica DMRXA/RF4/V automated universal microscope, and images were acquired with a Cooke SensiCam high performance digital camera using the Slidebook software package (Intelligent Imaging Innovations, Inc.) or a Zeiss LSM510 Confocal microscope. In all cases, images were exported to Adobe Photoshop for figure preparation.
Immunoblot Analysis
pREP3Xtea2+ cDNA was constructed by cloning the BamHI/SmaI 4.6-kb fragment from pSPORTtea2+cDNA into the BamH1 and SmaI sites of pREP3X ( cells transformed with these constructs were grown in EMM with appropriate supplements and 5 µg/ml thiamine (Sigma-Aldrich), and cells were washed three times with thiamine-free medium then grown overnight in thiamine-free medium. Cells were harvested, and protein extracts prepared by vortexing cells with glass beads in sample buffer.
Western blot analysis was performed as described in
Construction of Tea2-GFP Homologous Integration Strain
Tea2 was tagged with GFP at the COOH terminus as described in
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Results |
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Isolation of the Kinesin-like Gene, tea2+, by PCR and Phenotype Rescue
To understand the roles of klps in S. pombe, we carried out a PCR screen using degenerate primers to highly conserved regions in the motor domain of the kinesin superfamily. Two new S. pombe klps were identified, klp3+ (to be discussed elsewhere and
The PCR-generated clone also was used to map klp4+ by hybridization to a cosmid filter of the S. pombe genome (
In an independent, parallel series of experiments, we were investigating the localization of Tea1p in tea2-1 cells and found that Tea1p was mostly delocalized from the cell tips compared with wild-type cells and was found along the microtubules and in the cytoplasm (data not shown). This result suggests that Tea2p might be required to transport Tea1p to the cell tips. To investigate this possibility, tea2+ was mapped by positional cloning to cosmid c1604 (described in Materials and Methods). This cosmid was subcloned, and the 14T plasmid, a subclone capable of rescuing the tea2-1 morphology defects, was used for further analyses.
The similarity of the phenotypes of the knockout of klp4+ (described below) and mutant alleles of tea2+ ( phenotype (Table 2), and the plasmids 14B and 14H (Fig 1 A) also rescued both the klp4
phenotype (Table 2) and the tea2-1 phenotype. However, neither the deletion nor the tea2-1 mutant was rescued by the 5' truncation construct, 14X, that lacks 896 bp of the klp4+ ORF but leaves the downstream ORF intact (Fig 1 A). The 14T plasmid was also integrated into the genome, and the site of integration was genetically mapped to the tea2+ locus (described in Materials and Methods). In addition, the region corresponding to the klp4+ ORF was sequenced in DNA isolated from tea2-1 cells and shown to have a serine to phenylalanine transition at aa 384. This serine residue is in the motor domain and is a highly conserved aa found in nearly all klps.
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These results establish that tea2+ and klp4+ encode the same gene; from this point on, the gene will be referred to as tea2+ and its protein product as Tea2p.
Characterization of the tea2+ Transcript and Protein Product
Sequence analysis of the genomic and cDNA clones indicated that tea2+ potentially encodes a 628-aa protein that is expressed from a transcript of at least 4.6 kb and that contains 2.6 kb of 3' sequence. This 3' region contains a second ORF of 658 aa (accession nos. AL034433 and PID g4007771). Because this is an unusual structure for an S. pombe gene, we sought additional evidence for the gene structure of tea2+. The region corresponding to the tea2+ ORF hybridized to a 5-kb transcript on Northern blots (Fig 1 B, probe 1). Northern blot analyses using probes further 3' indicate that the
5-kb tea2+ transcript extends in this direction, and that a second transcript of 2.5 kb is present in this region (Fig 1 B, probes 2 and 3). This smaller transcript presumably codes for the ORF in this region that is predicted from the genomic sequence.
The junction between the two ORFs was confirmed by RT-PCR performed on RNA from wild-type cells. Primer B (Fig 1 A) at the predicted 5' end of the downstream gene was used for the reverse transcriptase reaction, and this primer in combination with primer A corresponding to the 3' end of the tea2+ ORF was used for PCR. An RT-PCR product of 315 nucleotides was produced, indicating that this region is uninterrupted by introns (not shown).
Analysis of the protein product further supports the proposed genomic structure of the region. Affinity-purified antibodies generated to the COOH-terminal region of Tea2p reacted with an 70-kD protein in wild-type cells, which was absent in cells deleted for the tea2+ ORF (Fig 1 C). Furthermore, tea2
cells expressing just the tea2+ ORF or the entire tea2+ cDNA under the control of the inducible nmt+ promoter produced a protein of the expected size for Tea2p (Fig 1 D).
Finally, the 2.6-kb 3' region of the tea2+ transcript is not essential for rescue of the mutant phenotype. A multicopy plasmid containing the tea2+ ORF, 14H, rescued the phenotype of both tea2 and tea2-1 cells (Fig 1 A; Table 2).
Sequence Comparison Analysis
Sequence searches using the motor domain of Tea2p revealed that of all the klps that have been characterized (beyond mere identification in a genome project), it is most similar to Saccharomyces cerevisiae Kip2p. Direct comparison of the motor domains, using the BestFit program from the GCG sequence analysis package, demonstrated that Tea2p and Kip2p are 58% similar and 51% identical over 332 aa. The motor domains of both proteins lie roughly in the middle of the proteins: the Kip2p motor domain extends from residues 97 to 500 within the 706-aa protein, and the Tea2p motor domain runs from residues 129 to 467 within the 628-aa protein. Outside the motor domain, the sequences are 35% similar and 26% identical over an 83-aa stretch in the NH2-terminal region and 46% similar and 32% identical over an 87-aa stretch in the COOH-terminal region (Fig 1 E). In the COOH terminus, Tea2p is predicted to contain one or two coiled coil regions of 2841 aa, depending on the matrix employed and whether the weighting option was used (
An alignment containing Tea2p, Kip2p, and 41 other kinesin family members was analyzed with the phylogenetic program PAUP (version 4.0), assuming maximum parsimony and using a heuristic search method with stepwise addition (described in Materials and Methods). This analysis revealed that of 100 bootstrap replicas, 93 grouped Kip2p, Tea2p, and CaKrp together (Fig 1 F; CaKrp is a klp identified by the Candida albicans genome project). A value of >90 strongly supports a phylogenetic relationship on statistical grounds (
Defects in Cell Morphology and in the Microtubule Cytoskeleton
To investigate further the cellular roles of Tea2p in S. pombe, a deletion allele was constructed by replacing the tea2+ ORF with his3+. Transformants were screened by PCR, and homologous integration was confirmed by Southern blot analysis (not shown). At 32°C, tea2 cells grow at rates similar to wild-type cells. These cells were examined by DIC microscopy to see if the deletion had an effect on the morphology of the cells. Cultures of exponentially growing cells contain
18% (n = 117) obviously bent cells, whereas wild-type cells were generally straight cylinders, 0% bent (n = 138; Fig 2A and Fig B). At 37°C, tea2
cells grew more slowly than wild-type cells, and a high percentage of T shaped cells were seen in the culture (up to 9%). These defects in cell morphology are similar to the tea2-1 mutant (
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Because defects in cell shape may be related to defects in the cytoskeleton and because tea2 mutant alleles have short cytoplasmic microtubules ( cells (Fig 3). Exponentially growing cells were stained with antibodies to tubulin, and the cytoplasmic microtubule network was found to be severely reduced (Fig 3). The defects appeared to be more severe when the cells were fixed with aldehyde rather than methanol, perhaps because of a difference in microtubule stability that is reflected by sensitivity to fixation. Astral microtubules were examined in tea2
cells using a tubulin-gfp construct (
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It seemed possible that a transition from a phase of nongrowth to a phase of growth might involve an extensive reestablishment of cell polarity and therefore necessitate a relocalization of tip-defining components. This possibility was supported by an observation made during the cloning of tea2+: tea2-1 cells had a more severe phenotype upon recovery from nutrient starvation. In addition, colonies of tea2 cells had variable percentages of T-shaped cells, perhaps caused by nutrient variations in the colony (Fig 2 D). To investigate these observations in more detail, we examined polarity reestablishment in tea2
cells as they emerged from stationary phase at 32°C. After tea2
cells were grown to saturation in liquid rich medium and then diluted into fresh medium, 75% (n = 700) acquired a T-shaped morphology. (Hundreds of wild-type cells examined all maintained their cylindical shape upon exit from stationary phase.) To more fully examine this defect in tea2
cells, 107 individual cells were followed by DIC microscopy through the first few divisions after release from stationary phase (Fig 4). 63 of the cells developed a T shape, 7 developed an L shape, and 6 developed other abnormal morphologies, whereas 31 developed relatively normally. T-shaped cells were tracked through their second division, and 36/39 of these cells grew again from the same ectopic site in the next division (Fig 4). In contrast, 34/34 of the normal shaped cells produced from the first division of the T-shaped cells underwent a normal subsequent division (Fig 4). This lineage analysis suggests that upon exit from stationary phase, a cell that intiates growth from an ectopic site generally continues to use that ectopic site in the subsequent division. Furthermore, once a cell acquires a nonbranched morphology (i.e., the daughter cell formed from the base of the T), this cell is able to maintain a relatively normal morphology in the following divisions. This latter point is further supported by the absence of T-shaped cells in exponentially growing cultures at 32°C.
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Enhancement of Morphological Defects with Increases in Cell Length
The short cytoplasmic microtubules in tea2 cells were generally clustered around the nucleus. This arrangement of the cytoskeleton might be especially detrimental in long cells because they may require a more extensive microtubule transport system for tip specification. To test this idea, the phenotype of tea2
in genetic backgrounds that result in long cells was examined. Entry into mitosis is delayed in cdc25-22 cells even at permissive temperature, so these cells are 54% longer than wild-type cells at the time of division (
cdc25-22 cells grown at permissive temperature formed microcolonies of very long and often branched cells (Fig 2, CF), indicating that the extra length of cdc25-22 cells cannot be tolerated in a tea2
background. This interpretation was supported by similar observations in diploid cells, which are 85% longer than haploid cells (
cells grew poorly; they are very unstable, haploidize at a high frequency, and are often bent or branched (Fig 2 G).
Examination of Essential Functions among klps
To investigate redundancy for essential functions of tea2+ and other klps, double, triple, and quadruple mutants were constructed with pkl1 (
(C. Troxell and J.R. McIntosh, personal communication), klp3
(described in Materials and Methods and in
. All possible mutant combinations were constructed. Deletions were monitored by the auxotrophic markers used to delete each gene and by colony PCR using primers specific for each deletion. All combinations were viable at 32°C. To test for temperature sensitivity, each strain was streaked on a YES agar plate and grown at 32°C. These plates were replica plated to EMM agar with appropriate supplements and YES agar plates, and were incubated at 20°C, 25°C, 32°C, and 35.5°C. All mutant combinations were able to grow at these temperatures, suggesting that there are no redundancies of essential functions between Tea2p and these other klps.
Localization of Tea2p
The cellular localization of Tea2p was determined by fusing the endogenous tea2+ at its 3' end with the gene for GFP by homologous recombination. Exponentially growing cells were examined by epifluorescence microscopy, and Tea2p-GFP was seen concentrated at the cell tips with some fluorescence throughout the cytoplasm, particularly as cytoplasmic dots (Fig 5 A). Because the fluorescence from Tea2p-GFP was faint, some cells were fixed and stained with antibodies to GFP in an effort to enhance the signal (Fig 5, BE). As in live cells, Tea2p-GFP was seen at the cell tips, but the signal to noise ratio of the overall cytoplasmic pattern was enhanced in fixed cells. Punctate staining throughout the cytoplasm was observed in these cells. This is likely to be a combined result of signal enhancement, due to the use of antibodies, and some delocalization caused by fixation. Costaining with antibodies to GFP and microtubules revealed that the most intense cytoplasmic dots generally colocalized with the interphase microtubules and were sometimes at the microtubules' ends (Fig 5, BE). In mitotic cells, Tea2p-GFP was less concentrated at the cell tips (Fig 5 D, cell with arrow).
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The localization of the GFP tagged allele was confirmed using antibodies generated against a fusion protein containing GST and the stalk/tail region of Tea2p. The resulting immune sera were affinity purified against a second fusion protein that contained the Tea2p stalk/tail region tagged with six histidines, and the purified antibodies were used to examine the localization of Tea2p in exponentially growing cells. Staining of tea2 cells showed very faint cytoplasmic background fluorescence (Fig 5 F). In exponentially growing wild-type cells, Tea2p was detected at the cell tips and often at the end of cytoplasmic microtubules (Fig 5, GJ), whereas in mitotic cells Tea2p was less concentrated at the cell tips (not shown).
Because of the severity of the morphological defects observed as cells emerged from stationary phase, the localization of Tea2p was examined in stationary phase cells and in cells as they were released from growth arrest. In fixed cells, Tea2p was more concentrated at the cell tips in stationary phase cells and in cells released from stationary phase than in exponentially growing cultures (Fig 6). This could be a reflection of increased resistance to delocalization by fixation, as well as to a change in distribution. Both in stationary phase and in cells exiting stationary phase, noncell tip staining was often found to be coincident with microtubules or microtubule ends (Fig 6).
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Tea2p Dependence on Microtubules for Localization
To determine whether microtubules are required for Tea2p localization, the position of Tea2p was determined in the presence of the microtubule poison methyl 2-benzimidazolecarbamate (MBC). Because Tea2p is concentrated at the cell tips in cells exiting from stationary phase, this transition was used to characterize the need for microtubules for Tea2p tip localization. Wild-type cells were grown to stationary phase, diluted into fresh medium, and allowed to grow for 25 min. MBC (25 µg/ml) or DMSO (control cells) was then added to the culture, and the cells were further incubated with aliquots removed at 5, 8, and 20 min for staining with antibodies to Tea2p and microtubules. Although the cytoplasmic microtubule network was severely reduced at the 5- and 8-min time points, Tea2p remained concentrated at the cell tips (Fig 7; 8-min time point shown). By 20 min, Tea2p was no longer concentrated at the cell tips, but after the drug was washed out and the microtubules were allowed to repolymerize, Tea2p relocalized to the cell tips (Fig 7). These results suggest that microtubules are required for transporting Tea2p to the tip but not for the short-term maintenance of this localization. Treatment with the microtubule poison thiabendazole (TBZ) or cold shock also resulted in the delocalization of Tea2p (data not shown).
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Localization of Tea1p in tea2-1 and tea2 Cells and Tea2p in tea1
Cells
Tea1p is proposed to be an end marker that directs the growth machinery to the cell tip (, and wild-type cells were stained with antibodies to Tea1p (Fig 8, AD; wild-type and tea2
cells shown). In wild-type cells, Tea1p localized to the cell tips (Fig 8 A;
mutant cells, Tea1p localized primarily to the short cytoplasmic microtubules (Fig 8C and Fig D; tea2
shown). Finally, to investigate whether Tea1p had an effect on Tea2p localization, we examined the localization of Tea2p-GFP in a tea1
strain. Tea2p-GFP was still localized at the cell tips, but was more extended in distribution along the microtubules compared with a wild- type strain (compare Fig 8E and Fig F, with Fig 5).
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Discussion |
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Tea2p Affects Cellular Morphology through an Interaction with Microtubules
We have shown that tea2+ encodes a klp that is required to establish proper cellular morphology in the fission yeast. Mutant alleles of tea2+ ( cells. These shape abnormalities are most severe in long cells, either diploids or mutants that are longer than haploid wild-type cells, and in cells progressing from a phase of nongrowth to a phase of growth. These results suggest that the importance of microtubules for normal cell growth varies with cell length and growth stage. Tea2p localizes to the cell tips and often to the ends of cytoplasmic microtubules including microtubules that do not reach the cell tip; its localization at cell tips is dependent on cytoplasmic microtubules. Analysis of microtubule dynamics in wild-type cells suggests that microtubules extend from the cell center out to the cell tips, with the minus ends located near the nucleus and the plus ends at the cell tips (
The localization of Tea2p and the phenotype of tea2 and tea2-1 mutants are consistent with two mechanisms by which Tea2p might function: Tea2p may affect the length of microtubules through a direct interaction with the microtubules, or it could act indirectly by transporting one or more proteins to the plus end of microtubules, which in turn results in microtubule stabilization. In either case, because there is a high concentration of microtubule plus ends at the cell tips (
In the first model, Tea2p could act directly on the end of a microtubule to affect the rate of polymerization or depolymerization, or the frequency of rescue or catastrophy. Previous analyses have revealed that klps can affect the dynamic stability of microtubules in vitro (
In the second model, Tea2p would bind tip-specific protein(s) and transport them along microtubules to their plus ends. The cargo proteins could then modulate microtubule stability, promoting growth. As the microtubules elongate, by either the direct or indirect mechanism, they would be expected to reach the cell tip; interaction there with the cell cortex could provide additional regulation of the length and stability of the microtubule.
Comparison of Tea2p with S. cerevisiae klps
The analyses of klps in S. cerevisiae have provided a wealth of information about the roles of these enzymes in cell behavior, but none of the mutations in budding yeast has the effect described here for the deletion of tea2+ in fission yeast. This is likely to be due to the observation that S. cerevisiae, in contrast to S. pombe, does not require cytoplasmic microtubules for morphological decisions and development (
Possible Cargoes of Tea2p: Tea1p Requires Tea2p for Proper Localization
Tea2p may also transport proteins that help to define the cell's growing tip. Several proteins that are important for cell morphology are also localized to the cell tip including Tea1p and Pom1p ( cells appears normal (
Tea1p has been proposed to direct the cell growth machinery to the cell tip (
In the absence of Tea2p, Tea1p localizes along the short cytoplasmic microtubules characteristic of tea2 cells. Therefore, although Tea1p has an affinity for microtubules in the absence of Tea2p, proper localization of Tea1p to the cell tip requires Tea2p. One possibility is that Tea2p transports Tea1p along microtubules and deposits it at the cell tip. A second possibility is that Tea1p uses another microtubule-mediated mechanism to get to the tip of the cell, and the absence of a normal array of cytoplasmic microtubules in tea2
cells results in the mislocalization of Tea1p.
Tea1p may also have a direct effect on Tea2p localization. When tea1+ is deleted, Tea2p is distributed more broadly along the microtubules, possibly because it is moving more slowly or binding to microtubules less efficiently. Alternatively, Tea1p may be required for efficient anchoring of Tea2p to the cell tip. Interestingly, although both proteins require microtubules for tip-specific localization, they are able to remain at the cell tips for short periods in the absence of microtubules, suggesting that the requirement for microtubules is for transport but not for anchorage (Fig 7;
Our analyses of Tea2p and tea2 mutant cells provide new evidence for the role of microtubules in the proper positioning of the growth site in fission yeast. The involvement of the microtubule cytoskeleton in the control of cell shape is a widely observed phenomenon that is likely to have many conserved components. Determining whether the mechanism by which Tea2p functions is through the direct stabilization of microtubules or the transport of a microtubule-regulating complex will provide insight into the control of morphogenesis in S. pombe, and this mechanism may represent a more general function of klps in the morphology of eukaryotic cells.
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Footnotes |
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Address correspondence to Heidi Browning, 44 Lincoln's Inn Fields, Imperial Cancer Research Fund, London WC2A 3PX, England. Tel.: 44-020-7269-3276. Fax: 44-020-7269-3258. E-mail: browninh{at}icrf.icnet.uk
H. Browning's present address is Cell Cycle Laboratory, Imperial Cancer Research Fund, London, UK. J. Mata's present address is Developmental Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
1 Abbreviations used in this paper: aa, amino acid(s); cM, centimorgan; DIC, differential interference contrast; EMM, Edinburgh minimal medium; GFP, green fluorescence protein; klp, kinesin-like protein; MBC, methyl 2-benzimidazole-carbonate; ORF, open reading frame; RT-PCR, reverse transcription followed by PCR; TBZ, thiabendazole; YES, yeast extract plus supplements.
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Acknowledgements |
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The authors wish to thank Robert West, Ken Sawin, Takashi Toda, Paula Grissom, Katya Grishchuk, Cynthia Troxell, Alison Pidoux, Zac Cande, and Damian Brunner for plasmids, antibodies, strains, helpful suggestions, and critical reading of the manuscript. We thank Mark Winey for the generous use of his microscope, which was sponsored in part by Virginia and Mel Clark. We are grateful to Scott Kelley for help with the phylogenetic analysis. Yuming Han in the University of Colorado automated sequencing facility sequenced the tea2+ clones.
This work was supported by National Institutes of Health (NIH) grant GM-36663 to J.R. McIntosh and by the Imperial Cancer Research Fund. H. Browning was supported in part by NIH postdoctoral fellowship GM-17117 and by a postdoctoral fellowship from the International Agency for Research on Cancer.
Submitted: 27 March 2000
Revised: 16 August 2000
Accepted: 17 August 2000
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References |
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