This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Juvvadi, P. R.
Right arrow Articles by Steinbach, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Juvvadi, P. R.
Right arrow Articles by Steinbach, W. J.

 Previous Article  |  Next Article 

Eukaryotic Cell, September 2008, p. 1606-1610, Vol. 7, No. 9
1535-9778/08/$08.00+0     doi:10.1128/EC.00200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Calcineurin Localizes to the Hyphal Septum in Aspergillus fumigatus: Implications for Septum Formation and Conidiophore Development{triangledown} ,{dagger}

Praveen Rao Juvvadi,1 Jarrod R. Fortwendel,1 Nadthanan Pinchai,1 B. Zachary Perfect,1 Joseph Heitman,2,3,4 and William J. Steinbach1,2*

Department of Pediatrics, Duke University Medical Center, Durham, North Carolina,1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina,2 Department of Medicine, Duke University Medical Center, Durham, North Carolina,3 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina4

Received 18 June 2008/ Accepted 23 June 2008


arrow
ABSTRACT
 
A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus {Delta}cnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.


arrow
TEXT
 
Calcineurin, a Ca2+-calmodulin-dependent protein phosphatase (PP2B), regulates diverse processes, including morphogenesis, ion homeostasis, virulence, and stress responses in fungi (1, 3-5, 9, 12, 13, 16-18, 28). In Aspergillus fumigatus, calcineurin is required for hyphal growth, cell wall integrity, conidial morphology, PO4– transport, and pathogenicity via its downstream target, CrzA (2, 6, 20-24). However, the specific molecular mechanisms by which the calcineurin signal transduction pathway regulates hyphal extension or cell wall biosynthesis remain largely unknown.

To elucidate calcineurin roles in hyphal growth, its localization pattern was analyzed during growth by fusion to enhanced green fluorescent protein (EGFP). The A. fumigatus cnaA cDNA, encoding the calcineurin A catalytic subunit, was inserted into the pUCGH plasmid (a gift from Axel Brakhage) (14). The cnaA cDNA was cloned at the N terminus of egfp to obtain the cnaA-egfp fusion allele (pUCGH-cnaA) under the control of the synthetic otef promoter. The A. fumigatus {Delta}cnaA mutant was transformed with pUCGH-cnaA, as previously described (21), and transformants were selected by resistance to hygromycin B. After single spore isolation, Southern analysis indicated that one of the six transformants contained a single-copy cnaA-egfp cassette integrated ectopically, and we have designated that as the cnaA-egfp expression strain (Fig. 1A, lane 4).


Figure 1
View larger version (70K):
[in this window]
[in a new window]

  		FIG. 1. (A) Genomic DNA from wild-type (AF293), {Delta}cnaA mutant, and cnaA-egfp strains digested with HincII and probed with a digoxigenin-labeled 650-bp PCR fragment in the cnaA open reading frame. Lanes 1, 2, 3, and 4 indicate the digoxigenin-labeled marker ladder and DNA from the wild-type, {Delta}cnaA mutant, and cnaA-egfp strains, respectively. The difference in band size observed between the wild-type strain (1,156 bp; Fig. 1A, lane 2) and the cnaA-egfp strain (1,044 bp; lane 3) is due to the presence of an intron in the wild-type strain. (B) Radial growth on glucose minimal agar media (GMM) inoculated with 103 conidia and colony diameters measured of the wild-type, {Delta}cnaA mutant, and the cnaA-egfp strains over 96 h at 37°C. Experiments were performed in triplicate, and a representative picture of the growth assay is shown. (C) Wild-type and the cnaA-egfp strains were grown as mentioned in panel B in the presence of 20 ng of FK506/ml. (D) For microscopic observation, 103 conidia of the respective strains were inoculated in 100 µl of GMM liquid medium in the absence or presence of FK506 and grown for a period of 24 h. Scale bar, 20 µm.

Because the {Delta}cnaA mutant exhibits stunted hyphal growth (6, 21), we next examined whether the cnaA-egfp expression strain returned to wild-type growth. As shown in Fig. 1B, radial growth, quantified as previously described (21), indicated that cnaA-egfp expression complemented the cnaA deletion and restored the wild-type phenotype. The cnaA-egfp expression strain was also grown in the presence of FK506, a specific inhibitor of calcineurin, to reconfirm that hyphal growth was stunted after FK506 treatment in a similar manner to that of the wild-type strain (Fig. 1C, plate assay). Microscopic examination of the wild-type and cnaA-egfp expression strains grown in the presence of FK506 also revealed a highly branched phenotype similar to the {Delta}cnaA mutant (Fig. 1D, lower panel). Taken together, these results indicate that the cnaA-egfp construct is functional after placement in the {Delta}cnaA mutant.

Next, in order to observe the localization patterns of CnaA-EGFP, the cnaA-egfp expression strain was grown on slide cultures for 24 h. While fluorescence microscopy revealed a general distribution of the CnaA-EGFP fusion protein in the cytoplasm, dot-like structures were observed closer to the cell wall in conidia (Fig. 2A and B) and at the hyphal tips (Fig. 2D) Very interestingly, the CnaA-EGFP fusion protein highly concentrated and localized on either side of the septa (Fig. 2G [see inset image]). The fusion protein localized both at the newly formed septa and the septa of the older hyphal compartments (Fig. 2C, E, F, and G). We noted that the CnaA-EGFP dot-like structures moved and concentrated in the middle of the septum (Fig. 2F [see inset image indicated by arrowheads]). Three-dimensional imaging of the septum showed a disk-like appearance of CnaA-EGFP localization around the septal pore (see the figure in the supplemental material). In addition, the CnaA-EGFP fusion protein localized to dot-like structures (resembling cortical patches) during the formation of the conidiophore. As shown in Fig. 3A and B, the dot-like structures seemed to move into the vesicle and localize at junctions between the vesicle and phialides in a mature conidiophore (Fig. 3D). The CnaA-EGFP spots outlined the points of formation of a new septum as well (Fig. 3B, arrowheads). Transformation of A. fumigatus with the pUCGH plasmid alone showed only cytoplasmic localization of EGFP (14).


Figure 2
View larger version (82K):
[in this window]
[in a new window]

  		FIG. 2. Localization of CnaA-EGFP during growth of A. fumigatus. The cnaA-egfp strain was grown in 100 µl of GMM liquid medium on coverslips for 24 h and observed by fluorescence microscopy. (A) Distribution of CnaA-EGFP in dormant conidia at 0 h of growth. (B) Swollen conidium at 3 h of growth. CnaA-EGFP is localized in small vesicular structures at the region of germ tube formation. (C) Germling at 6 h of growth showing a newly formed septum to which CnaA-EGFP is localized. (D) The CnaA-EGFP is localized to the hyphal tip in dot-like structures. (E) Middle regions of hypha showing mature septa with CnaA-EGFP. (F) CnaA-EGFP fusion protein in dot-like structures seen closely associated with the central region of the septum. (G) Vesicle showing the localization of CnaA-EGFP at its septum. Scale bar, 10 µm.


Figure 3
View larger version (54K):
[in this window]
[in a new window]

  		FIG. 3. Localization of CnaA-EGFP during conidiophore formation. The cnaA-egfp strain was grown in 100 µl of GMM liquid medium on coverslips for 24 h and observed by fluorescence microscopy. (A) Dot-like cortical patches in a newly forming conidiophore (indicated by arrows). (B) Dot-like structures decorating a newly forming septum (indicated by arrowheads) in a conidiophore. (C) CnaA-EGFP is localized to the septum in the conidiophore. (D) CnaA-EGFP is seen localized to junctions between the vesicle and phialides in a mature conidiophore. Scale bar, 10 µm.

Because the CnaA-EGFP localized to points of phialide formation, we next examined whether conidiophore development was impaired in the {Delta}cnaA mutant and, as well, upon treatment of the wild-type and cnaA-egfp expression strains with FK506. In contrast to conidiophores observed in the cnaA-egfp expression and wild-type strains (Fig. 1D, upper panel), {Delta}cnaA mutant or FK506 treated cells showed a complete absence of conidiophores, indicating calcineurin is required for conidiophore formation (Fig. 1D, lower panel). In addition, hyphae treated with FK506 showed several septa dividing smaller compartments, in comparison to the untreated wild-type strain that showed septa at regular intervals dividing large compartments (data not shown), indicating that calcineurin may be required for regular hyphal extension and septation and a possible reason for the overall blunted growth following calcineurin inhibition. In Schizosaccharomyces pombe it was reported that calcineurin localized at the septa, and its deletion resulted in thick and incomplete septation (15). Interestingly, a recent study in Aspergillus nidulans has implicated a role for the protein phosphatase 1 (BIMG) in septum formation (10). However, in contrast to our results, BIMG-GFP only transiently localized to the septum.

Microtubules and actin filaments are involved in vesicle transport in filamentous fungi (8). The presence of calcineurin in dot-like structures at the hyphal tips, as cortical patches during conidiophore formation, at junctions of phialide formation, and at the septa, prompted us to examine whether the microtubular network or actin filaments were contributing to calcineurin localization. To test this hypothesis, the CnaA-EGFP expression strain was grown for 16 h and then treated with nocodazole (to inhibit microtubules) or cytochalasin A (to inhibit actin polymerization) for an additional 8 h. While the nocodazole-treated sample did not show any mislocalization of CnaA-EGFP (data not shown), the cytochalasin A-treated sample showed a complete cytosolic redistribution of CnaA-EGFP (Fig. 4C and D), indicating that the transport of calcineurin to the septum is an actin-dependent process. Treatment of hyphae grown for 16, 20, and 22 h with cytochalasin A for additional time periods of 1, 4, and 2 h, respectively, also mislocalized CnaA-EGFP from the septa (data not shown). These data also revealed that actin may be required for not only formation of septa but also the maintenance of CnaA-EGFP at completed septa (septa that were formed before the addition of cytochalasin A).


Figure 4
View larger version (96K):
[in this window]
[in a new window]

  		FIG. 4. Effect of cytochalasin A on the localization of CnaA-EGFP. The cnaA-egfp strain was grown in 100 µl of GMM liquid medium for 16 h and then cytochalasin A (80 µg/ml [panels C and D]) was added. The cultures were grown for an additional 8 h and examined by fluorescence microscopy. (A and B) Control cultures with CnaA-EGFP localized to septa in the absence of the inhibitor; (C and D) cytosolic localization of CnaA-EGFP due to actin inhibition. Scale bar, 10 µm.

In addition to reports on localization of chitin synthases at the septum (11, 25), two recent reports on A. nidulans PkcA::GFP (26) and SwoM::GFP (27), also involved in the cell wall integrity pathway, showed localization patterns similar to that observed in our study. Interestingly, calmodulin, which binds and activates calcineurin, has also been implicated in the organization of actin cytoskeleton (7, 19, 29). Future directions will include an examination of the interaction of calcineurin with other proteins mediating cell wall biosynthesis. It would be premature at this stage of analysis to ascribe a detailed mechanism for calcineurin localization and functions at the septum and in conidiophore development. Our future studies will address the questions of how and why calcineurin localizes at the septum, since it is currently unclear whether cnaA mRNA localizes at the septum and is translated locally or is transported to the septum via its interacting proteins.


arrow
ACKNOWLEDGMENTS
 
W.J.S. is supported by an NIH/NIAID K08 A1061149 award, a Basic Science Faculty Development grant from the American Society for Transplantation, and a Children's Miracle Network grant.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC 27710. Phone: (919) 681-1504. Fax: (919) 684-8902. E-mail: stein022{at}mc.duke.edu Back

{triangledown} Published ahead of print on 7 July 2008. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back


arrow
REFERENCES
 
    1
  1. Bader, T., K. Schroppel, S. Bentink, N. Agabian, G. Kohler, and J. Morschhauser. 2006. Role of calcineurin in stress resistance, morphogenesis, and virulence of a Candida albicans wild-type strain. Infect. Immun. 74:4366-4369.[Abstract/Free Full Text]
    2. 2
  2. Cramer, R. A., Jr., B. Z. Perfect, N. Pinchai, S. Park, D. S. Perlin, Y. G. Asfaw, J. Heitman, J. R. Perfect, and W. J. Steinbach. 2008. Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot. Cell 7:1085-1097.[Abstract/Free Full Text]
    3. 3
  3. Cruz, M. C., D. S. Fox, and J. Heitman. 2001. Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J. 20:1020-1032.[CrossRef][Medline]
    4. 4
  4. Cruz, M. C., A. L. Goldstein, J. R. Blankenship, M. Del Poeta, D. Davis, M. E. Cardenas, J. R. Perfect, J. H. McCusker, and J. Heitman. 2002. Calcineurin is essential for survival during membrane stress in Candida albicans. EMBO J. 21:546-559.[CrossRef][Medline]
    5. 5
  5. Cyert, M. S. 2003. Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochem. Biophys. Res. Commun. 311:1143-1150.[CrossRef][Medline]
    6. 6
  6. da Silva Ferreira, M. E., T. Heinekamp, A. Härtl, A. A. Brakhage, C. P. Semighini, S. D. Harris, M. Savoldi, P. F. de Gouvêa, M. H. de Souza Goldman, and G. H. Goldman. 2007. Functional characterization of the Aspergillus fumigatus calcineurin. Fungal Genet. Biol. 44:219-230.[CrossRef][Medline]
    7. 7
  7. Desrivières, S., F. T. Cooke, H. Morales-Johansson, P. J. Parker, and M. N. Hall. 2002. Calmodulin controls organization of the actin cytoskeleton via regulation of phosphatidylinositol (4,5)-bisphosphate synthesis in Saccharomyces cerevisiae. Biochem. J. 366:945-951.[Medline]
    8. 8
  8. Fischer, R., N. Zekert, and N. Takeshita. 2008. Polarized growth in fungi-interplay between the cytoskeleton, positional markers and membrane domains. Mol. Microbiol. 68:813-826.[CrossRef][Medline]
    9. 9
  9. Fox, D. S., and J. Heitman. 2002. Good fungi gone bad: the corruption of calcineurin. Bioessays 24:894-903.[CrossRef][Medline]
    10. 10
  10. Fox, H., P. C. Hickey, J. M. Fernández-Abalos, P. Lunness, N. D. Read, and J. H. Doonan. 2002. Dynamic distribution of BIMG(PP1) in living hyphae of Aspergillus indicates a novel role in septum formation. Mol. Microbiol. 45:1219-1230.[CrossRef][Medline]
    11. 11
  11. Ichinomiya, M., E. Yamada, S. Yamashita, A. Ohta, and H. Horiuchi. 2005. Class I and class II chitin synthases are involved in septum formation in the filamentous fungus Aspergillus nidulans. Eukaryot. Cell 4:1125-1136.[Abstract/Free Full Text]
    12. 12
  12. Juvvadi, P. R., Y. Kuroki, M. Arioka, H. Nakajima, and K. Kitamoto. 2003. Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation. Arch. Microbiol. 179:416-422.[Medline]
    13. 13
  13. Kraus, P. R., and J. Heitman. 2003. Coping with stress: calmodulin and calcineurin in model and pathogenic fungi. Biochem. Biophys. Res. Commun. 311:1151-1157.[CrossRef][Medline]
    14. 14
  14. Langfelder, K., B. Philippe, B. Jahn, J. P. Latgé, and A. A. Brakhage. 2001. Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein. Infect. Immun. 69:6411-6418.[Abstract/Free Full Text]
    15. 15
  15. Lu, Y., R. Sugiura, T. Yada, H. Cheng, S. O. Sio, H. Shuntoh, and T. Kuno. 2002. Calcineurin is implicated in the regulation of the septation initiation network in fission yeast. Genes Cells 7:1009-1019.[Abstract]
    16. 16
  16. Mendoza, I., F. J. Quintero, R. A. Bressan, P. M. Hasegawa, and J. M. Pardo. 1996. Activated calcineurin confers high tolerance to ion stress and alters the budding pattern and cell morphology of yeast cells. J. Biol. Chem. 271:23061-23067.[Abstract/Free Full Text]
    17. 17
  17. Miyakawa, T., and M. Mizunuma. 2007. Physiological roles of calcineurin in Saccharomyces cerevisiae with special emphasis on its roles in G2/M cell-cycle regulation. Biosci. Biotechnol. Biochem. 71:633-645.[CrossRef][Medline]
    18. 18
  18. Odom, A., S. Muir, E. Lim, D. L. Toffaletti, J. Perfect, and J. Heitman. 1997. Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J. 16:2576-2589.[CrossRef][Medline]
    19. 19
  19. Sekiya-Kawasaki, M., D. Botstein, and Y. Ohya. 1998. Identification of functional connections between calmodulin and the yeast actin cytoskeleton. Genetics 150:43-58.[Abstract/Free Full Text]
    20. 20
  20. Soriani, F. M., I. Malavazi, M. E. da Silva Ferreira, M. Savoldi, M. R. Von Zeska Kress, M. H. de Souza Goldman, O. Loss, E. Bignell, and G. H. Goldman. 2008. Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA. Mol. Microbiol. 67:1274-1291.[CrossRef][Medline]
    21. 21
  21. Steinbach, W. J., R. A. Cramer, B. Z. Perfect, Y. G. Asfaw, T. C. Sauer, L. K. Najvar, W. R. Kirkpatrick, T. F. Patterson, D. K. Benjamin, J. Heitman, and J. R. Perfect. 2006. Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5:1091-1103.[Abstract/Free Full Text]
    22. 22
  22. Steinbach, W. J., R. A. Cramer, B. Z. Perfect, C. Henn, K. Nielsen, J. Heitman, and J. R. Perfect. 2007. Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus. Antimicrob. Agents Chemother. 51:2979-2981.[Abstract/Free Full Text]
    23. 23
  23. Steinbach, W. J., J. L. Reedy, R. A. Cramer, Jr., J. R. Perfect, and J. Heitman. 2007. Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections. Nat. Rev. Microbiol. 5:418-430.[CrossRef][Medline]
    24. 24
  24. Stie, J., and D. Fox. 2008. Calcineurin regulation in fungi and beyond. Eukaryot. Cell 7:177-186.[Free Full Text]
    25. 25
  25. Takeshita, N., S. Yamashita, A. Ohta, and H. Horiuchi. 2006. Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth. Mol. Microbiol. 59:1380-1394.[CrossRef][Medline]
    26. 26
  26. Teepe, A. G., D. M. Loprete, Z. He, T. A. Hoggard, and T. W. Hill. 2007. The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans. Fungal Genet. Biol. 44:554-562.[CrossRef][Medline]
    27. 27
  27. Upadhyay, S., and B. D. Shaw. 2006. A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation. Fungal Genet. Biol. 43:739-751.[Medline]
    28. 28
  28. Yoshida, T., T. Toda, and M. Yanagida. 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]
    29. 29
  29. Yuan, J., G. X. Shi, Y. Shao, G. Dai, J. N. Wei, D. C. Chang, and C. J. Li. 2008. Calmodulin bound to stress fibers but not microtubules involves regulation of cell morphology and motility. Int. J. Biochem. Cell Biol. 40:284-293.[CrossRef][Medline]

Eukaryotic Cell, September 2008, p. 1606-1610, Vol. 7, No. 9
1535-9778/08/$08.00+0     doi:10.1128/EC.00200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Juvvadi, P. R.
Right arrow Articles by Steinbach, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Juvvadi, P. R.
Right arrow Articles by Steinbach, W. J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»