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Vol. 15, Issue 4, 1969-1980, April 2004
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Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Stra
e, D-35043 Marburg, Germany
Submitted September 17, 2003;
Revised December 22, 2003;
Accepted December 27, 2003
Monitoring Editor: David Drubin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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eca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca2+/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in eca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Ubiquitous effectors of Ca2+ signaling are Ca2+/calmodulin-dependent protein kinases (CaMKs) and the Ca2+/CaM-dependent phosphatase calcineurin, which act by modulating the phosphorylation state of several proteins. The Ca2+ signal terminates when Ca2+-ATPases and Ca2+/H+ exchangers transport Ca2+ out of the cell or sequester it into organelles, thereby reducing cytosolic Ca2+ concentration to basal levels. The endoplasmic reticulum (ER) is the major Ca2+ store in most eukaryotic cells and sarco/ER-resident Ca2+-ATPases (SERCA; East, 2000) are required to pump Ca2+ into the ER so as to maintain low resting levels of cytosolic Ca2+ and to activate Ca2+ binding chaperones that assist the folding and modification of secretory proteins (Corbett and Michalak, 2000).
There is a growing body of evidence demonstrating a requirement for Ca2+-dependent signaling during the infection or invasion phase in diverse parasites of plants and animals, such as Histoplasma capsulatum (Sebghati et al., 2000) and Cryptococcus neoformans (Fox and Heitman, 2002). In particular, recent work in fungal pathogens reveals that inhibiting the function of calcineurin and Ca2+/CaMKs has major effects on fungal growth and leads to a loss in virulence (Joseph and Means, 2000; Cruz et al., 2002). Comparable pathways implicating Ca2+ signaling in the regulation of growth cone extension and filopodial motility in mammalian cells have been described (Gomez and Spitzer, 1999), suggesting that the role of Ca2+ in regulating morphogenesis is of widespread relevance. A potential target of Ca2+ signaling is the cytoskeleton that supports polar growth and determines the morphology of the cell. CaMKs are thought to regulate the activity of actin-based myosin V (Karcher et al., 2001). In addition, it has been shown that altered calcium homeostasis affects morphology and microtubule (MT) dynamics in fission yeast (Facanha et al., 2002), and CaMKs might also be involved, because it was shown that they regulate MT dynamics by phosphorylation of the MT regulator stathmin (Gardin et al., 1997).
Here, we describe a role of calcium in morphogenesis of the corn smut fungus, Ustilago maydis. This model pathogen is amenable to molecular genetics, its genome is published, and it is perfectly suited to analyze fungal dimorphism and pathogenicity (Bölker, 2001). Furthermore, it deserves attention because it shares similarities with vertebrate systems in several aspects of its cell biology, including unexpected gene conservation with higher eukaryotes (Kojic et al., 2002), a highly dynamic MT cytoskeleton (Steinberg et al., 2001; Straube et al., 2003), and the existence of kinesin motors, such as Kif1A- and a conventional kinesin (Lehmler et al., 1997; Wedlich-Söldner et al., 2002b), which are also crucial for axonal transport (Hirokawa, 1998). Moreover, similar to vertebrate cells intracellular motility of endosomes and ER depends on MTs and associated dynein (Wedlich-Söldner et al., 2000, 2002a).
As an initial step toward elucidating the role of Ca2+ signaling in morphogenesis in this fungus, we have characterized eca1, which was isolated by complementing a temperature-sensitive (ts) mutant. We show that Eca1 represents a true SERCA that is required for proper growth and cell survival. At standard growth conditions, mutants show a defective cell morphology that most likely results from altered MT dynamics and organization. Detailed analysis of parameters of MT dynamic instability, as well as ER motility suggests an involvement of cytoplasmic dynein in the eca1 phenotype. Both the morphology phenotype and MT defect can be rescued by inhibition of CaM-kinases, whereas inhibition of the Ca2+-dependent phosphatase calcineurin aggravated the phenotype of eca1 mutants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Strains and Plasmids
All strains had the genetic background of FB1 (a1b1) or FB2 (a2b2; for details of plasmids and strains, see Table 1). In FB2Eca1 eca1 was deleted by a complete replacement with a phleomycin resistance cassette. For in vivo analysis of green fluorescent protein (GFP)-marked MTs, the a GFP-
-tubulin-encoding plasmid (potefGFPTub1; Table 1) was ectopically integrated into the succinate-dehydrogenase locus of strains FB1, FB2Eca1, and FB1Dyn2ts, resulting in FB1GT, FB1 and Dyn2tsGT, and FB2GT and FB2Eca1GT, respectively. For localization of U. maydis GFP-myosin V plasmid, pOG-Myo5 was introduced in FB2Myo5 (Weber et al., 2003) and FB2Eca1. The ER network in FB2Eca1 was visualized by integration of plasmid pERGFP (Wedlich-Söldner et al. 2002a), and colocalization studies were done in strain FB2EYEC that was derived from FB2 transformed with plasmids pEca1YFP and pERCFP. Strain FB2Eca1Dyn2ts was generated by replacing dyn2 by the temperature-sensitive allele dyn2ts. In this strain, potef-GFP-Tub1 was introduced, resulting in FB2Eca1Dyn2tsGT. For detection of intracellular calcium, a GFP-based Ca2+ probe (Nakai et al., 2001) was put under the control of the otef-promoter and ectopically integrated in FB2 and FB2Eca1 cells. All integrations were confirmed by Southern blot analysis, and transformants were checked for proper morphology and doubling time to minimize possible defects due to the integration. For functional complementation of yeast strain K616 (Cunningham and Fink, 1994), eca1 was expressed under the control of the gal1-10-promoter (pGEca1, derived from plasmid YEplac195; kindly provided by Dr. H. Ulrich, MPI Marburg, Germany).
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Growth Conditions
Strains were grown at 22 and 30°C in CM medium (Holliday, 1974) supplemented with 1% glucose (CM-G) or nitrate minimal medium supplemented with 5.6 mM Ca2+ (NM; Holliday, 1974) and without Ca2+ (NM/low Ca2+). Temperature shift experiments by using strains containing the dyn2ts-allele were done as described previously (Wedlich-Söldner et al., 2002a). For temperature shifts of eca1 mutant strains, 20 ml of liquid cultures was grown overnight in CM-G at 22°C to OD600 < 1, and 3-5 ml of this culture was supplemented with dimethyl sulfoxide, benomyl, KN-92, or KN-93 (Calbiochem, San Diego, CA) and shifted to 30°C in a water bath. Salt conditions were analyzed at 22°C. For low Ca2+ experiments, cells of 15-ml overnight cultures were washed twice in 15 ml of precooled water and resuspended in 5 ml of NM or NM/low Ca2+ medium, followed by incubation for 4 h at 30°C in culture flasks that were rinsed with ultrapure water. Colony growth of K616 derivatives was monitored on synthetic complete medium without uracil supplemented with 10 mM CaCl2 or 40 mM EGTA. Plate growth of U. maydis was analyzed on CM-G plates containing CaCl2 (1-1000 mM), EGTA (1-500 mM), LiCl (1-1000 mM), and NaCl (1-1000 mM). Inhibitor effects were analyzed on CM-G plates supplemented with the solvent dimethyl sulfoxide or ethanol, 0.5-3 µM benomyl, 10 µM cytochalasin D, 1-20 µM taxol, 1-10 µM tunicamycin, 1.5-3 mM dithiothreitol (DTT), 15-30 µg/ml cyclosporin A, and 1-4 µg/ml FK506 (kindly donated by Fujisawa GmbH, Munich, Germany). Unless otherwise noted, chemicals were purchased at Sigma Chemicals.
Southern, Northern, and Western Analysis
DNA isolation from U. maydis and transformation procedures were carried out as described previously (Krüger et al., 1998). RNA was isolated from strains grown in liquid culture for 3 h and prepared as described previously (Krüger et al., 1998). A 2005-bp SacII-KpnI fragment of eca1 was used as a probe. Western analysis was done as described previously (Straube et al., 2001) by using a monoclonal GFP antibody (Roche Diagnostics, Mannheim, Germany) and horseradish peroxidase-coupled anti-mouse IgG (Promega, Madison, WI).
Sequence Analysis
Phylogenetic dendograms were constructed using ClustalX and MEGA 2.1 (http://www.megasoftware.net) by using the minimum evolution or maximum parsimony algorithms and gap deletion option. Then 500 replicates were used for bootstrap support. Domain and motif analysis was carried out using BLAST (http://www.ncbi.nlm.nih.gov/blast/) and SMART (http://smart.embl-heidelberg.de) servers. Transmembrane helices were predicted with ISREC (http://www.ch.embnet.org). The genome of U. maydis was accessed at http://www.genome.wi.mit.edu/annotation/fungi/ustilago_maydis/index.html).
Light Microscopy and Image Processing
Microscopic analysis was performed using an Axioplan II microscope (Carl Zeiss, Jena, Germany). Frames were taken with a cooled charge-coupled device camera (C4742-95; Hamamatsu, Bridgewater, NJ). Epifluorescence of GFP was observed using the standard fluorescein isothiocyanate filter sets. Colocalization of YFP and CFP was analyzed with specific filter sets (YFP: BP500/20, FT515, and BP535/30; and CFP: BP436, FT455, and BP480-500). Quantification and image processing, including adjustment of brightness, contrast, and gamma values, was performed with ImageProPlus (Media Cybernetics, Gleichen, Germany), MetaMorph (Universal Imaging, Downing-town, PA), and Photoshop (Adobe Systems, Mountain View, CA).
Detection of Cytosolic Ca2+
Cells expressing a GFP-based calcium probe (Nakai et al., 2001) were grown in CM-G at 22°C. Cells were shifted incubated for 3 h at 30°C in a water bath, and samples were observed at 50% of lamp intensity (Atto Arc; Carl Zeiss) by using the standard fluorescein isothiocyanate filters and a cooled charge-coupled device camera (Coolsnap HQ; Photometrics, Tucson, AZ). To minimize the risk of artifacts due to oxygen depletion or radiation, only two to three cells per preparation were observed without embedding in agarose. Average cytoplasmic signal intensities were measured and corrected by the background noise and the autofluorescence of FB2 cells by using MetaMorph (Universal Imaging).
Quantification of Morphology Defects, ER Motility, and Microtubule Dynamics
For microscopic observation, cells from logarithmic cultures were embedded in 1% low melt agarose. Unless stated, morphology of control and eca1 mutant cells was analyzed after 4-5 h at 30°C. Cell morphology was considered "abnormal" if cells were multiple budded, branched, or irregularly shaped. For each experiment, at least 100 cells were analyzed. ER motility and MT dynamics was measured as described previously (Steinberg et al., 2001; Wedlich-Söldner et al., 2002a). All analysis was done using digital sequences of 30-60 frames that were taken with an exposure time of 500-1000 ms per frame at 40-60% lamp intensity. Microscopic preparations were observed no longer than 15 min to prevent defects due to oxygen depletion. All measurements were done using ImageProPlus (Universal Imaging). Statistical analysis by two-tailed t test at = 0.05 was carried out using Prism (GraphPad Software Inc., San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Figure 1A). The gene product Eca1 is predicted to contain 10 transmembrane domains and all motifs characteristic of P-type ATPases, including those implied in nucleotide binding and phosphorylation (Evans and Williams, 1998), as well as the evolutionary conserved Ca2+ binding sites (Clarke et al., 1989; Figure 1B). In addition, Eca1 contains an N-terminal ER targeting pentapeptide motif found in SERCAs (Magyar and Varadi, 1990) and a canonical dilysine C-terminal ER retention motif (KKQL; Andersson et al., 1999). To verify the predicted localization of Eca1 in the ER, we expressed an Eca1-GFP fusion protein in the eca1 mutant strain FB1Eca1ts. The fusion protein rescued the growth defect of the mutant and localized to a peripheral network and the nuclear envelope, a characteristic distribution of the ER (our unpublished data). To confirm this notion, we then coexpressed in wild-type cells a fusion protein of Eca1 and yellow fluorescent protein (Eca1-YFP; Figure 1C1) and an ER-targeted cyan fluorescent derivative (ER-CFP; Figure 1C2) that has been previously shown to localize to the ER in U. maydis (Wedlich-Söldner et al., 2002a). Both Eca1-YFP and ER-CFP colocalized in all areas (Figure 1C3), thereby confirming that Eca1 resides in the ER.
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Eca1 Functions in Ca2+ Homeostasis
Sequencing of the mutated eca1 allele in FB1Eca1ts, revealed a single point mutation (A to T) at nucleotide +1576 that changed residue K526 to a stop codon, therefore making it likely that, regardless of temperature, only a nonfunctional Eca1 protein is expressed in this mutant. Consequently, we deleted the entire eca1 open reading frame. At 30°C, which is close to the optimum temperature of U. maydis, growth of the resulting strain FB2Eca1 was reduced (Figure 3A; wt, wild-type control; eca1, eca1 mutant) and cell morphology of mutants was heavily impaired (see below), whereas morphology and the ability to form colonies was restored at 22°C and abolished at 34°C (Figure 2A).
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eca1 cells by using a GFP-based Ca2+ probe that exhibits fluorescence upon binding to Ca2+ (Nakai et al., 2001). Western analysis showed that the reporter gene was expressed at similar levels in reference strain FB2CMP (Figure 3A) and eca1 mutant strain FB2Eca1CMP (eca1CMP). A quantitative analysis on the single cell level revealed a faint Ca2+ signal at 30°C in reference cells that was slightly decreased at 22°C (Figure 3B, not significantly different from 22°C, p = 0.1146; for example, see 3C1 and 3C2). In contrast, Ca2+ levels were strongly elevated in eca1 cells at 30°C and significantly decreased at 22°C (Figure 3B, 3C2, and 3C4; p < 0.0001), indicating that Eca1 is required to maintain normal cytosolic Ca2+ concentrations. In some cases, growth of eca1 mutants was not restored at 22°C, and this coincided with high Ca2+ levels, indicating that abnormal morphology and elevated Ca2+ levels are related (Figure 3B).
To confirm a role of Eca1 in Ca2+ transport, we complemented the yeast mutant strain K616 (Cunningham and Fink, 1994), an approach that was successfully used to confirm Ca2+ transport activity of SERCA pumps before (Sze et al., 2000). In this yeast mutant, two Ca2+ ATPases, Pmc1p and Pmr1p, are deleted, resulting in a requirement for external Ca2+ supplies. On plates supplemented with Ca2+ the mutant K616 transformed with empty vector (control; Figure 3D) or vector carrying the U. maydis eca1 gene (pGEca1; Figure 3D) formed colonies. However, growth of the control strain was abolished on a medium depleted for Ca2+ (control, 40 mM EGTA; Figure 3D), whereas expression of eca1 restored viability of K616 under these conditions (Figure 3D). Eca1-YFP expressed in K616 localized to the nuclear envelope and a peripheral network (arrow in Figure 3E), which suggests that Eca1 complements the defects of K616 by its Ca2+-pumping activity at the ER.
Because these data argue for a role of Eca1 in Ca2+ transport into the ER, where Ca2+ions are needed for ER-dependent folding of secretory proteins, we tested this ER function in eca1 cells. We generated ER stress by treatment with tunicamycin (2.5 µg/ml) and DTT (2 mM), which affect N-glycosylation and folding reactions in ER. Wild-type cells still grew under these conditions, but both agents inhibited growth of eca1 already at 22°C (Figure 2B). This hypersensitivity indicates that ER-based protein processing is compromised in eca1 mutants, which might be the cause of the lethal phenotype at 34°C. Growth of eca1 strains was also sensitive to EGTA (5 mM; Figure 2C), but neither high concentrations of Ca2+ (250 mM) nor Mn2+ (10 mM) inhibited colony formation, although most eca1 cells showed a pronounced morphology defect under these conditions (see below). Finally, Northern analysis indicated that eca1 was expressed at similar levels under various stress conditions, and a slight increase was only found at high temperature (p = 0.018) or 50 mM extracellular Ca2+ (p = 0.015; Figure 2D). In summary, these data strongly indicate that Eca1 regulates Ca2+ homeostasis by pumping cytosolic Ca2+ into the ER.
Eca1 Is Required for Interphase MT Organization and Polar Growth
At 30°C, eca1 cells showed growth at both poles and formed multiple septa (Figure 4A2, 4 h at 30°C; septa indicated by arrows). This resulted in large cell aggregates with rounded and apolar cells (Figure 4A3, 12 h at 30°C). Under these conditions, wild-type cells were unaffected (Figure 4A3, inset, and 4C). Low temperature (22°C) restored morphology of eca1 cells (Figure 4A1, for wild-type control, see inset in Figure 4A3). Interestingly, this temperature phenotype was dependent on external Ca2+ concentration, because low extracellular Ca2+ suppressed the temperature-dependent eca1 phenotype (Figure 4B, compare with 4A2; 4C; 4 h at 30°C in NM with low Ca2+). In addition, morphology of eca1 cells was more sensitive to high extracellular Ca2+ levels (250 mM; Figure 4C), which also affected the shape of wild-type cells but to a much smaller extent. Thus, Eca1 is required to maintain normal cell shape at 30°C and during Ca2+ stress.
Morphology of fungal cells is based on the cytoskeleton. Therefore, we speculated that the Ca2+-dependent phenotype of FB2Eca1 was due to defects in cytoskeletal organization or function. Therefore, we first monitored the effect of cytoskeleton inhibitors on plate growth of wild-type control and eca1. In the presence of 10 µM of the actin inhibitor cytochalasin D, no difference between control and eca1 mutants was observed (Figure 4D), and this coincided with normal actin patch distribution in eca1 mutants at restrictive conditions (our unpublished data). Furthermore, in FB2eca1 transport along the actin cytoskeleton was apparently not impaired, because a functional GFP-myosin V fusion protein that requires F-actin for polar localization at the growth region (Weber et al., 2003) was correctly positioned at the cell poles (Figure 4E, 3 h 30°C). This suggested that elevated Ca2+ levels did not severely affect the actomyosin system. In contrast, eca1 mutant were more sensitive to the MT stabilizer taxol (5 µM) and less sensitive to low doses of destablizer benomyl (1 µM; Figure 4D). Moreover, 2.5 µM benomyl suppressed most of the temperature-dependent morphology defects of eca1 cells (Figure 4F1 and 4F2; 4 h at 30°C, compare with 4A2), indicating that growth and morphology defects of FB2eca1 are mainly due to unusually stable MTs. This notion was further supported by the observation that GFP- tubulin-labeled MTs (Steinberg et al., 2001) in eca1 cells were much longer and irregular arranged (Figure 4G, eca1; see also 5D2) as in reference strain FB2GT (Figure 4G, control, 4 h at 30°C). Consistent with the restorative effect of lower temperature on morphology no obvious differences between the mutant and control cells were found at 22°C (Figure 4G). These results were confirmed by indirect immunofluorescence by using anti-tubulin antibodies in strains FB2 and FB2Eca1, indicating that they are not due to expression of GFP- tubulin (our unpublished data). Interestingly, disturbed MT patterns were observed in wild-type cells exposed to high extracellular Ca2+ (250 mM; Figure 4H), suggesting that aberrant MTs and cell shape are generally associated with increased Ca2+ levels. Together, these results argue for an effect of high cytosolic Ca2+ levels on morphogenesis by influencing MT stability in eca1.
Ca2+ Signaling Is Involved in the Phenotype of eca1 Mutants
The effects of high Ca2+ levels on MTs might be mediated by effectors of Ca2+ signaling, Ca2+/CaM dependent-kinases, and the phosphatase calcineurin. Similar to eca1, calcineurin mutants in fungi show poor viability under ER stress conditions (Bonilla et al., 2002; Cruz et al., 2002). In addition, these mutants are sensitive to cations such as Li2+ and Na+. Consequently, we checked plate growth of eca1 mutants on LiCl (7.5 mM) and NaCl (0.5 M). No growth inhibition was observed, and eca1 cells grew even slightly better on LiCl, indicating that calcineurin was not inhibited in these cells (Figure 5A). Moreover, exposure of eca1 mutants to the specific calcineurin inhibitors cyclosporin A (15 µg/ml) or FK506 (4 µg/ml; Ho et al., 1996), led to a significant reduction in growth at 30°C (Figure 5A), whereas wild-type cells were not affected. In other words, the dephosphorylation activity of calcineurin is crucial for survival of eca1 on plates.
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In contrast, the inhibition of CaMK by the potent inhibitor KN-93 (Sumi et al., 1991) had a positive effect on eca1 cells. Treatment with 60 µM KN-93, but not with its inactive structural analogue KN-92, restored near-normal morphology to eca1 mutants at 30°C (Figure 5B1 and 5B2), whereas this treatment did not affect morphology of wild-type cells (Figure 5B3). Restoration of morphology by KN-93 treatment was accompanied by the formation of nearly normal interphase MT arrays (Figure 5C1) in 
70% of all cells after 3 h at 30°C, whereas MT arrays in KN-92-treated cells were disordered and resembled the characteristic eca1 phenotype (Figure 5C2). Thus, altered Ca2+-homeostasis in eca1 mutants seems to increase CaMK activity, which in turn deregulates MT dynamics and results in morphology defects.
eca1 and Dynein Mutants Share Virtually Identical Alterations in Microtubule Dynamics
The defect in MT organization in FB2eca1 resembled that of dynein mutants (Straube et al., 2001). Therefore, we examined the relationship between eca1 and dynein effects on MT dynamics. We first analyzed the role of dynein in MT organization by using a temperature-sensitive allele of the dynein heavy chain (dyn2ts; Wedlich-Söldner et al. 2002a). At permissive temperature, MTs in dyn2ts and control cells were indistinguishable (Figure 6, A and B, 22°C), but inactivation of dynein at 29-30°C led to much longer, curved, and disorganized MTs (Figure 6B, 4-6 h at 29°C). This phenotype was reminiscent of eca1 mutants (compare Figures 6B and 4H or 5C2). In both mutants, MTs seemed less dynamic and more often growing MTs were observed (Figure 6C, dynts; 6D, eca1).
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). To gain deeper insight into the MT phenotype, we measured these parameters of dynamic instability in eca1 and dyn2ts mutant strains and compared the results to their reference strains. In both mutants, the velocity of elongation and shrinkage of MTs was almost unaltered, but in eca1 and dyn2ts cells rescue rate was threefold increased and catastrophe rates were twofold lowered (Figure 6E and Table 2). Thus, it seems that shrinking MTs more often restart growth, whereas elongating MTs less often switch to depolymerization, explaining the abnormally long MTs in dyn2ts and eca1 mutants. Inhibition of CaMKs by KN-93 resulted in almost normal MT organization of eca1 at 30°C (see above), and MT elongation and shrinkage rates, as well as rescue frequencies of eca1 mutants were restored to near normal levels (Figure 6F and Table 2). In contrast, MT defects in dynein mutants were not restored by the CaMK inhibitor KN-93 (Figure 6B), indicating that KN-93 acts upstream of the dynein complex in regulation of MT stability.
eca1 Mutants Are Defective in Dynein-dependent ER Motility
To gain further support for the hypothesis that the defects of eca1 mutants are due to altered dynein activity, we examined the effect of deletion of eca1 on another dynein-dependent process. The peripheral tubular ER network in U. maydis is highly dynamic (Figure 7A1, arrow; see inset for higher magnification) and this motility is solely dynein-dependent (Wedlich-Söldner et al., 2002a). To analyze ER organization and motility in eca1 cells, we generated a strain that expressed an ER-targeted GFP fusion protein (FB2Eca1EG). At 22°C, the ER was localized at the cell periphery of both eca1 and control cells (our unpublished data) and directed tubule motility in both strains was indistinguishable (Figure 7C; eca1: 4.12 ± 0.36 events/h x µm2; reference: 4.59 ± 0.60 events/h x µm2, n > 35 cells from three experiments; p = 0.3288). Temperature shift had no effect on the ER in control cells, but under these conditions eca1 mutants contained an irregular network of ER tubules that were no longer located at the cell periphery (Figure 7, B and D). At elevated temperature, ER motility in control cells increased, but directed ER tubule motion in eca1 was significantly reduced (Figure 7, B and C; eca1: 1.40 ± 0.14 events/h x µm2, reference: 8.94 ± 0.23 events/h x µm2; n = 3 experiments and >38 cells; p > 0.0001). This defect in ER-motility in eca1 corresponds well with that of dynein mutants (Figure 7C; not significantly different, p = 0.1340, values for dyn2ts from Wedlich-Söldner et al., 2002a). To analyze minor effects on ER motility, we did a quantitative comparison of digital images at two time points by using the software package MetaMorph. Again, we measured significantly less total ER displacement in eca1 than in control cells (p = 0.0059; 10-13 measurements, 28 cells). Finally, we set out to rescue the ER motility defect by the CaMK inhibitor KN-93. Although this CaMK inhibitor had no effect on ER in control cells at 30°C, KN-93 treatment led to fragmentation and disorganization of the ER networks in eca1 mutants that did not allow a study of motility. Interestingly, temperature shift led to a nuclear distribution defect in eca1. Control cells contained a single nucleus in the cell center that is detectable by ER-GFP in the nuclear envelope (Figure 7A, arrowhead). However, eca1 at 30°C often contained numerous nuclei (Figure 7D, overnight 30°C; see also arrow in Figure 7B) that were also detected by 4,6-diamidino-2-phenylindole staining of DNA (Figure 7E, arrowheads; maximum-Z-axis projection of cells grown overnight at 30°C). Such a defect is characteristic of dynein mutants (Straube et al., 2001), again supporting the notion that dynein function is impaired in eca1. Together, these results demonstrate that dynein-dependent motility of ER tubules is significantly impaired in eca1 mutants, again indicating that elevated Ca2+ levels in eca1 affect dynein activity.
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Dynein and Eca1 Double Mutants Show Synthetic Growth Phenotypes, but No Increase in MT Defects
To gain deeper insights in the functional relationship between Eca1 and dynein, we generated strain FB2Eca1Dyn2ts. Shifting this double mutant to 30°C simultaneously inactivated dynein and increased cytosolic Ca2+. Assuming that Eca1 and dynein act in the same pathway, inactivation of both should not increase the phenotype of the individual mutants. Growing mutants at 30°C in liquid culture resulted in a more severe morphological defect of the double mutant (Figure 8A, 11 h). In addition, at semipermissive conditions (Figure 8B, 28°C) the double mutant was clearly more impaired in plate growth, whereas growth of all mutants was the same at permissive temperature (Figure 8B, 22°C). However, mutations in eca1 and dynein showed no synthetic effect on MT organization (Figure 8C), and the alterations in MT dynamics in the double mutant were comparable with that of the eca1 single mutant strain (Figure 8D; changes in parameters compared with strain FB2Eca1GT). Therefore, we conclude that Eca1 and dynein are in the same pathway to regulate MT stability, which further supports the notion, an effect of Ca2+ signaling on MTs via the dynein complex. However, the increased growth defects of the double mutant argue for additional targets of Ca2+ signaling in morphology and cell separation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), and elevated levels of cytosolic calcium in eca1 mutants implicate a function for Eca1 in Ca2+ transport into the ER. However, eca1 cells grow almost normally at 22°C, suggesting that other Ca2+ pumps partially compensate for the loss of Eca1 at this temperature. In addition to the vacuolar and Golgi-localized Ca2+-ATPases PMC1 and PMR1 in budding yeast (Cunningham and Fink, 1996), both budding and fission yeasts contain the V-subtype of ER-resident P-ATPases (Cronin et al., 2002; Facanha et al., 2002), which are also implicated in Ca2+ homeostasis. Surveys of the Ustilago genome (see MATERIALS AND METHODS) indicate that an orthologue of these proteins exists (47.7% identity and 64% similarity >1244 amino acids to Cta4p from Schizosaccharomyces pombe). Elucidating the role of this additional putative Ca2+ pump and their functional interplay with Eca1 will be a challenge for the nearer future.
Deregulated Ca2+ Signaling Is Responsible for the Defect of eca1 Mutants
Eca1 is dispensable at low temperature but is required to maintain morphology at 30°C. This indicates that a rise in temperature creates conditions under which transport of Ca2+ into the ER becomes crucial for the cell. In addition, treatment with tunicamycin and DTT, which targets protein modification and folding in the ER (Bonilla et al., 2002), is lethal to eca1 cells. A likely explanation for these observations is that higher temperatures increase the need for Ca2+-dependent protein folding in the ER and that Eca1 activity is required to provide Ca2+ as a cofactor for ER-resident chaperones (Corbett and Michalak, 2000). Obviously, this raises the possibility that misfolding of secretory proteins in the ER cause the observed morphology defect. However, the morphology phenotype of eca1 can be suppressed by lowering extracellular Ca2+ and by specific inhibition of cytosolic CaMK by using KN-93 (Sumi et al., 1991). Moreover, in eca1 cytosolic Ca2+ is increased. This implies that temperature rise induces influx of extracellular Ca2+ that cannot be stored in the ER in the absence of Eca1. In this model, this transport defect elevates cytosolic Ca2+ levels and leads to an increase of CaMK activity (Figure 9). The high sensitivity of eca1 mutants to inhibition of calcineurin further supports this notion, because it indicates that this Ca2+-dependent phosphatase counteracts the hyperactive CaMKs, thereby enabling eca1 to survive at 30°C. A link between calcineurin and CaMK-dependent signaling and cell morphology was also reported in fission yeast (Yoshida et al., 1994; Rasmussen, 2000) and in neurons (Chang et al., 1995). The accumulation of misfolded proteins in the ER of higher eukaryotes induces an entry of external Ca2+ into the cell (Putney et al., 2001), and SERCA activity is required to shuffle this Ca2+ into the ER (East, 2000). Therefore, we consider it most likely that similar mechanisms exist in U. maydis and that Eca1 is a key component facilitating survival and proper morphology under certain stress conditions.
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Morphology Defects of eca1 Cells Are Due to Disordered MTs
Fungal growth and morphology depend on transport along the cytoskeleton, and a growing body of evidence indicates that MTs play a key role in this (Seiler et al., 1997; Sawin and Nurse, 1998; Wedlich-Söldner et al., 2000). eca1 mutants show severe defects in polar growth and cytokinesis, suggesting that cytoskeleton-based growth processes are affected. In agreement, the abnormal eca1 cells contained much longer and disorganized MTs, a defect that might be a consequence of deregulated dynamic instability of MTs. On the other hand, it is important to consider that elevated Ca2+ levels almost certainly affect numerous other cytoskeletal targets. For example, Myo5, a class V myosin involved in polar growth in U. maydis (Weber et al., 2003) contains a PEST site for Ca2+-regulated proteolysis and a putative CaMK phosphorylation site that is implied in Ca2+-dependent regulation of organelle traffic (Karcher et al., 2001). A functional GFP-myosinV fusion protein localized to the growing ends of eca1 cells, suggesting that Myo5 is not degraded and migrates along F-actin toward growth sites. Moreover, actin patch distribution was normal in eca1 (our unpublished data), indicating that the actin cytoskeleton is not severely affected by the deletion of eca1. On the other hand, high Ca2+ could interfere with other myosins or affect binding of Myo5 to secretory vesicles, thereby resulting in growth defects in eca1. However, the eca1 growth and morphology defect was significantly restored by benomyl, which is thought to destabilize MTs at low concentrations (Willins et al., 1995). Thus, altered morphology of eca1 cells is most likely a consequence of altered MT dynamics, although it cannot be excluded that additional but so far undetected defects add to the observed phenotype.
Is Dynein a Target of Ca2+ Signaling to Regulate MT Length and Organization?
MT-associated proteins control dynamic instability (Walczak, 2000), and these components are potential targets of CaMKs (Gardin et al., 1997). Evidence exists for a role of dynein in the control of MT dynamics in fungi (Carminati and Stearns, 1997; Han et al., 2001), and our data presented here support a role of dynein in modifying MT stability. Surprisingly, both dynein and eca1 mutants show strikingly similar defects in MT dynamics. In both strains, MTs were longer, curved, and this is accompanied by a threefold increase in rescue rates and a twofold decrease in the frequency of MT catastrophe events. It is important to note that the altered parameters in MT number and dynamics are specific signatures for the dynein phenotype, because mutants in other MT-associated proteins or motors have different effects on MT dynamics (Walczak, 2000; our unpublished data). Therefore, the striking similarity between both the eca1 and dynein mutant phenotype is remarkable and, together with the restorative effect of CaMK inhibition on MT organization, suggest a link between Ca2+ signaling and dynein activity. In addition, the inactivation of dynein in eca1 mutants (strain FB2Deca1Dyn2tsGT) did not significantly increase the defects in MT dynamics. In other words, MT defects in both eca1 and dyn2ts background do not add in the double mutant, which supports our model of Eca1 acting on MT stability via dynein (Figure 9). This notion is further supported by the analysis of ER motility, a process that solely depends on cytoplasmic dynein in U. maydis (Wedlich-Söldner et al., 2002a). In both eca1 and dyn2ts mutants, ER tubule motility was inhibited to the same extent, whereas ER motility was not affected by disruption of F-actin or deletion of kinesin (Wedlich-Söldner et al. 2002a). Therefore, dynein activity is apparently impaired in eca1 mutants, and this might be a result of CaMK-dependent phosphorylation. Consistent with this hypothesis, it was recently shown that dynein activity in flagellar axonemes is modulated by CaMKs (Smith, 2002). Although Ustilago dynein heavy chain contains several potential CaMK phosphorylation sites, we consider it unlikely that the heavy chain is directly regulated by CaMKs. A good candidate for this regulation is the conserved 8-kDa dynein light chain that associates with calmodulin (Yang et al., 2001). However, details of the composition of the dynein complex in U. maydis are presently lacking.
In summary, our data are consistent with the notion that deletion of eca1 that encodes a SERCA leads to deregulated Ca2+-dependent signaling, which in turn affects MT dynamics via impaired dynein activity in U. maydis. SERCA activity is indispensable for Notch receptor and secretory protein transport in Drosophila melanogaster (Periz and Fortini, 1999), and defects in calcium homeostasis affect MT catastrophe rates and growth of fission yeast (Facanha et al., 2002). Therefore, the described link between Ca2+ signaling and MT-dependent exocytosis might be of general importance for eukaryotic cells.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: CaM, calmodulin; CaMK, Ca2+-calmodulin-dependent kinase; ER, endoplasmic reticulum; CFP, cyan fluorescent protein; GFP, green fluorescent protein; MT, microtubule; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; ts, temperature sensitive; YFP, yellow fluorescent protein.
* Present address: Hanulova 1, 84101 Bratislava, Slovakia 
Present address: Wellcome Trust Centre for Cell Biology, ICMB, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, Scotland, UK
Present address: Institut für Zellbiologie, LMU, Schillerstr. 42, D-80336 München, Germany.
Corresponding author. E-mail address: gero.steinberg{at}staff.unimarburg.de.
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|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Bannuett, F., and Herskowitz, I. (1989). Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proc. Natl. Acad. Sci. USA 86, 5878-5882.
Benito, B., Garciadeblas, B., and Rodriguez-Navarro, A. (2000). Molecular cloning of the calcium and sodium ATPases in Neurospora crassa. Mol. Microbiol. 35, 1079-1088.[CrossRef][Medline]
Bölker, M. (2001). Ustilago maydis - a valuable model system for the study of fungal dimorphism and virulence. Microbiology 147, 1395-1401.
Bonilla, M., Nastase, K.K., and Cunningham, K.W. (2002). Essential role of calcineurin in response to endoplasmic reticulum stress. EMBO J. 21, 2343-2353.[CrossRef][Medline]
Carminati, J.L., and Stearns, T. (1997). Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex. J. Cell Biol. 138, 629-641.
Chang, H.Y., Takei, K., Sydor, L.A., Born, T., Rusnak, F., and Jay, D.G. (1995). Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin. Nature 376, 686-690.[CrossRef][Medline]
Clarke, D.M., Loo, T.W., Inesi, G., and MacLennan, D.H. (1989). Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature 339, 476-478.[CrossRef][Medline]
Corbett, E.F., and Michalak, M. (2000). Calcium, a signalling molecule in the endoplasmic reticulum? Trends Biochem. Sci. 25, 307-311.[CrossRef][Medline]
Cronin, S.R., Rao, R., and Hampton, R.Y. (2002). Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis. J. Cell Biol. 157, 1017-1028.
Cruz, M.C., Goldstein, A.L., Blankenship, J.R., Del Poeta, M., Davis, D., Cardenas, M.E., Perfect, J.R., McCusker, J.H., and Heitman, J. (2002). Calcineurin is essential for survival during membrane stress in Candida albicans. EMBO J. 21, 546-559.[CrossRef][Medline]
Cunningham, K.W., and Fink, G.R. (1994). Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J. Cell Biol. 124, 351-363.
Cunningham, K.W., and Fink, G.R. (1996). Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 2226-2237.[Abstract]
East, J.M. (2000). Sarco(endo)plasmic reticulum calcium pumps: recent advances in our understanding of structure/function and biology. Mol. Membr. Biol. 17, 189-200.[CrossRef][Medline]
Evans, D.E., and Williams, L.E. (1998). P-type calcium ATPases in higher plants - biochemical, molecular and functional properties. Biochim. Biophys. Acta 1376, 1-25.[Medline]
Facanha, A.L., Appelgren, H., Tabish, M., Okorokov, L., and Ekwall, K. (2002). The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics. J. Cell Biol. 157, 1029-1039.
Fox, D.S., and J. Heitman. (2002) Good fungi gone bad: the corruption of calcineurin. Bioessays 24, 894-903.[CrossRef][Medline]
Gardin, H.M., Marklund, U., Larsson, N., Chatila, T.A., and Gullberg, M. (1997). Regulation of microtubule dynamics by Ca2+/Calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18. Mol. Cell. Biol., 17, 3459-3467.[Abstract]
Gomez, T.M., and Spitzer, N.C. (1999). In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397, 350-355.[CrossRef][Medline]
Han, G., Liu, B., Zhang, J., Zuo, W., Morris, N.R., and Xiang, X. (2001). The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr. Biol. 11, 719-724.[CrossRef][Medline]
Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519-526.
Ho, S., Clipstone, N., Timmermann, L., Northrop, J., Graef, I., Fiorentino, D., Nourse, J., and Crabtree, G.R. (1996). The mechanism of action of cyclosporin A and FK506. Clin. Immunol. Immunopathol. 80, S40-S45.[CrossRef][Medline]
Holliday, R. (1974). Ustilago maydis. In: Handbook of Genetics, ed. R.C. King, New YorK: Plenum Press.
Joseph, J.D., and Means, A.R. (2000). Identification and characterization of two Ca2+/CaM-dependent protein kinases required for normal nuclear division in Aspergillus nidulans. J. Biol. Chem. 275, 38230-38238.
Karcher, R.L., Roland, J.T., Zappacosta, F., Huddleston, M.J., Annan, R.S., Carr, S.A., and Gelfand, V.I. (2001). Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II. Science 293, 1317-1320.
Kojic, M., Kostrub, C.F., Buchman, A.R., and Holloman, W.K. (2002). BRCA2 homolog required for proficiency in DNA repair, recombination, and genome stability in Ustilago maydis. Mol. Cell 10, 683-691.[CrossRef][Medline]
Krüger, J., Loubradou, G., Regenfelder, E., Hartmann, A., and Kahmann, R. (1998). Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis. Mol. Gen. Genet. 260, 193-198.[CrossRef][Medline]
Lehmler, C., Steinberg, G., Snetselaar, K.M., Schliwa, M., Kahmann, R., and Bolker, M. (1997). Identification of a motor protein required for filamentous growth in Ustilago maydis. Embo J. 16, 3464-3473.[CrossRef][Medline]
Magyar, A., and Varadi, A. (1990). Molecular cloning and chromosomal localization of a sarco/endoplasmic reticulum-type Ca2(+)-ATPase of Drosophila melanogaster. Biochem. Biophys. Res. Commun. 173, 872-877.[CrossRef][Medline]
Nakai, J., Ohkura, M., and Imoto, K. (2001). A high signal-to-noise Ca(2+. probe composed of a single green fluorescent protein. Nat. Biotechnol. 19, 137-141.[CrossRef][Medline]
Periz, G., and Fortini, M.E. (1999). Ca2+-ATPase function is required for intracellular trafficking of the Notch receptor in Drosophila. EMBO J. 18, 5983-5993.[CrossRef][Medline]
Putney, J.W., Jr., Broad, L.M., Braun, F.J., Lievremont, J.P., and Bird, G.S. (2001). Mechanisms of capacitative calcium entry. J. Cell Sci. 114, 2223-2229.
Rasmussen, C.D. (2000). Cloning of a calmodulin kinase I homologue from Schizosaccharomyces pombe. J. Biol. Chem. 275, 685-690.
Sawin, K.E., and Nurse, P. (1998). Regulation of cell polarity by microtubules in fission yeast. J. Cell Biol. 142, 457-471.
Sebghati, T.S., Engle, J.T., and Goldman, W.E. (2000). Intracellular parasitism by Histoplasma capsulatum: fungal virulence and calcium dependence. Science 290, 1368-1372.
Seiler, S., Nargang, F.E., Steinberg, G., and Schliwa, M. (1997). Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa. EMBO J. 16, 3025-3034.[CrossRef][Medline]
Smith, E.F. (2002). Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase. Mol. Biol. Cell. 13, 3303-3313.
Steinberg, G., Wedlich-Soldner, R., Brill, M., and Schulz, I. (2001). Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity. J. Cell Sci. 114, 609-622.[Abstract]
Straube, A., Brill, M., Oakley, B.R., Horio, T., and Steinberg, G. (2003). Microtubule organization requires cell cycle dependent nucleation at dispersed cytoplasmic sites, polar and perinuclear MTOCs in the plant pathogen Ustilago maydis. Mol. Biol. Cell 14, 642-657.
Straube, A., Enard, W., Berner, A., Wedlich-Soldner, R., Kahmann, R., and Steinberg, G. (2001). A split motor domain in a cytoplasmic dynein. EMBO J. 20, 5091-5100.[CrossRef][Medline]
Stull, J.T. (2001). Ca2+-dependent cell signalling through calmodulin-activated protein phosphatase and protein kinases minireview series. J. Biol. Chem. 276, 2311-2312.
Sumi, M., Kiuchi, K., Ishikawa, T., Ishii, A., Hagiwara, M., Nagatsu, T., and Hidaka, H. (1991). The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem. Biophys. Res. Commun. 181, 968-975.[CrossRef][Medline]
Sze, H., Liang, F., Hwang, I., Curran, A.C., and Harper, J.F. (2000). Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 433-462.[CrossRef][Medline]
Verde, F., Dogterom, M., Stelzer, E., Karsenti, E., and Leibler, S. (1992). Control of microtubule dynamics by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J. Cell Biol. 118, 1097-1108.
Walczak, C.E. (2000). Microtubule dynamics and tubulin interacting proteins. Curr. Opin. Cell Biol. 12, 52-56.[CrossRef][Medline]
Weber, I., Gruber, C., and Steinberg, G. (2003). A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis. Plant Cell 15, 2826-2842.
Wedlich-Söldner, R., Bölker, M., Kahmann, R., and Steinberg, G. (2000). A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J. 19, 1974-1986.[CrossRef][Medline]
Wedlich-Söldner, R., Schulz, I., Straube, A., and Steinberg, G. (2002a). Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis. Mol. Biol. Cell. 13, 965-977.
Wedlich-Söldner, R., Straube, A., Friedrich, M.W., and Steinberg, G. (2002b). A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis. EMBO J. 21, 2946-2957.[CrossRef][Medline]
Willins, D.A., Xiang, X., and Morris, N.J. (1995). An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans. Genetics 141, 1287-1298.[Abstract]
Yang, P., Diener, D.R., Rosenbaum, J.L., and Sale, W.S. (2001). Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes. J. Cell Biol. 153, 1315-1326.
Yoshida, T., Toda, T., and Yanagida, M. (1994). A calcineurin-like gene ppb1+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J. Cell Sci. 107, 1725-1735.[Abstract]
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