QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02414

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Crampin, H. Articles by Sudbery, P. Search for Related Content PubMed PubMed Citation Articles by Crampin, H. Articles by Sudbery, P. Social Bookmarking                         What's this?

Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae

¹ Department of Molecular Biology and Biotechnology, Sheffield University, Western Bank, Sheffield, S10 2TN, UK
² University of Minnesota, Department of Genetics, Cell Biology and Development, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
³ Department of Pediatrics, University of Minnesota, MMC 39, 420 Delaware Street SE, Minneapolis, MN 55455, USA
⁴ Department of Microbiology, University of Minnesota, 6-170 MCB Building, 420 Washington Avenue SE, Minneapolis, MN 55455, USA

View larger version (50K): [in a new window]   Fig. 1. Mlc1 and FM4-64 colocalise to a Spitzenkörper-like structure at hyphal tips. (A) Colocalisation of Mlc1-YFP (green) and FM4-64 (red). Images shown are projections of the Z-stack of deconvolved images. In the merged panel, there is extensive overlap between Mlc1-YFP and FM4-64. (B) Detail of Mlc1 localisation at hyphal tips. Top left, An Mlc1 spot and crescent are visible. Right panels, the Mlc1-spot is present either at the tip (top) or just behind the tip (bottom). (C) Optical sections at 200 nm intervals through an individual germ tube tip. The crescent comes into focus at a different optical plane compared to the spot (arrow). Hyphal tips are outlined in panels B and C. (D) Hyphal germ tubes of an Mlc1-YFP-expressing strain were stained with filipin to reveal the plasma membrane. A 3D model was generated from the deconvolved Z-stack using Volocity® software. Mlc1-YFP (green) forms a 3D ball located behind the cell membrane (blue). (E) Localisation of Mlc1 (red) and the septin, Cdc11 (green), revealed by immunocytofluorescence using polyclonal antisera to the respective S. cerevisiae proteins. Specificity of the Cdc11 antibody has been described elsewhere (Sudbery, 2001). The anti-Mlc1 antibody recognised a single band in western blots that was the same size as S. cerevisiae Mlc1 (data not shown). (F) The shape of a hypha can be predicted by the VSC model according to the distance of the spot from the hyphal tip. An Mlc1-YFP expressing strain was induced to form hyphae and cell walls were stained with Concanavalin A-Texas Red. Measurements were made of external width at various distances from the tip. These were compared with the predicted widths at these points made by the Fungus Simulator program version 4 according to the distance of the centre of the Mlc1-YFP spot to the outside of the hyphal tip. Bar, 5 µm (A); 1 µm (B,C,F); 0.5 µm (D); 2 µm (E).

View larger version (89K): [in a new window]   Fig. 2. Localisation of Bni1-YFP, Spa2-YFP and Bud6-GFP. (A) The fluorescence from Bni1-YFP merged with the DIC image of a germ tube. A single spot is observed just behind the tip (arrow). (B) Z-stack projection of hyphal cells expressing Spa2-YFP, which can be seen in all cells to form crescents or caps, with areas of higher intensity staining. (C) Localisation of Bud6-GFP, which predominantly forms a crescent, although in some cells a spot of higher fluorescence is observed (arrow). (D) Partial colocalisation of Spa2-YFP and Mlc1-CFP in hyphal cells. (E) Partial colocalisation of Spa2-YFP and FM4-64 in hyphal cells. Arrow indicates the tip that is enlarged in the model below. The model was constructed using the model-building module in the SoftworxTM suite. Spa2-YFP is shown as a green wireframe, FM4-64 is shown in solid red. The Spa2 cap covers the solid body of FM4-64. Bar, 5 µm (A); 1 µm (B,E); 2 µm (C,D).

View larger version (80K): [in a new window]   Fig. 3. Localisation of YFP-Cdc42. (A-C) Cells expressing YFP-Cdc42 from the PCK1 promoter were grown in hyphal-inducing conditions and stained with FM4-64. The hyphal tip identified by the arrow in panel C is shown at greater magnification in panels D-F. Arrow in D indicates the subapical spot of YFP-Cdc42, which colocalises with FM4-64 resulting in the yellow spot in the merged image (F). Bar, 5 µm (A-C); 1 µm (D-F).

View larger version (82K): [in a new window]   Fig. 4. Mlc1 localisation to the Spitzenkörper depends on Spa2 and Bud6. (A,B) spa2/spa2 or bud6/bud6 strains, both expressing Mlc1-YFP, were grown under hyphal-inducing conditions. In both mutants, no spot of Mlc1-YFP is present, but a crescent remains. (C-E) DIC images of mutant hyphal cells (C,D) compared to wild-type hyphal cells (E). In both mutants, the hyphae are broader and more kinked than the wild-type hyphae. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 5. Organisation of the Spitzenkörper depends on microtubules. (A) A strain expressing Tub1-YFP was induced to form hyphal germ tubes from unbudded stationary phase yeast cells. Long microtubules are visible along with spots (arrow), which we presume are spindle pole bodies (SPBs). (B) After 60 minutes, 0.1 mg/ml MBC was added and the incubation was continued for a further 10 minutes. Microtubules have disappeared but the SPBs remain (arrow). (C) An Mlc1-YFP expressing strain was also treated with MBC in a parallel experiment. The cells were stained with FM4-64 after 10 minutes of treatment with MBC. The spot of FM4-64 staining has disappeared. (D) Localisation of Mlc1-YFP in the same cells shown in C. The spot of Mlc1-YFP has disappeared but a surface crescent remains in some cells. Bar, 5 µm.

View larger version (35K): [in a new window]   Fig. 6. The role of actin cables in Spitzenkörper integrity and polarised growth. Unbudded yeast cells expressing Mlc1-YFP were induced to form hyphae, stained with Concanavalin A (Con A) conjugated to either Alexa 488 or Texas Red and treated with 10 µm Cytochalasin A (CA) and as described in the flow chart. The protocol results in the formation of young germ tubes in which the cell walls fluoresce green. Cell walls formed after CA treatment are not stained with Con A-A488 (green) but are stained with Con A-TR (red). (A-C) A CA-treated hypha. (D-F) An untreated control hypha. CA treatment results in swollen tips that are stained red but not green. Thus, the swelling arises from new isotropic growth, not from swelling of a pre-existing hyphal tip. Mlc1-YFP, which is also visible in the green channel, disappears from the tip of CA-treated cells, but remains in the typical Spitzenkörper organisation in untreated cells (D, arrow; F, inset). An Mlc1 cytokinetic ring persists in the CA-treated cells (arrow in C). The response of hyphal germ tubes to CA treatment was uniform in the culture and the images shown are typical. Bar, 5 µm.

View larger version (55K): [in a new window]   Fig. 7. Mlc1, Spa2 and FM4-64 colocalise to a surface crescent structure in pseudohyphal buds. (A-F) Pseudohyphae were induced in strains carrying the indicated fusions to YFP or stained with FM4-64. Cells were stained with Calcofluor white (A-C) and with filipin (D). The images shown are a projection of the deconvolved Z-stack, except C, which is a single layer. Numbers in panels A and B refer to cells discussed in the text. (D) A pseudohyphal cell expressing Mlc1-YFP and stained with filipin. Left, deconvolved image; right, model generated with the SoftworxTM suite. (E and F) Localisation of Spa2-YFP and FM4-64, respectively, in pseudohyphae. Arrows in F indicate crescents of FM4-64 staining at the tips of pseudohyphal buds. (G) Mlc1-YFP-expressing pseudohyphal cells were sampled at various times after inoculation and fixed and stained with DAPI. Cells were categorised into bins of bud length and characterised according to whether Mlc1-YFP formed a ball or a crescent at the tip or was not present at all. In addition, cells that contained two nuclei were categorised as having completed mitosis. Note that the categories at each bud length do not add up to 100% as cells can be in more than one category. For example, a cell can be categorised as having no Mlc1 at the tip as well as having a cytokinetic ring. Each bin contains at least 100 cells.

View larger version (26K): [in a new window]   Fig. 8. Mlc1 and Spa2 localisation in yeast cells. Images were recorded from exponentially growing yeast cells expressing Mlc1-YFP (A) or Spa2-YFP (B). Mlc1 localises to a surface crescent in small buds, but to a ring at the mother-bud neck in large cells.

View larger version (43K): [in a new window]   Fig. 9. Mlc1 localisation is cell cycle-dependent in yeast and pseudohyphae. (A) Time-lapse video recorded from a pseudohyphal cell coexpressing Nop1-CFP and Mlc1-YFP. Numbers in each frame indicate time elapsed in minutes since inoculation of unbudded cells into fresh medium. The video may be viewed in supplementary material. (B,C) The axial ratio (length/width) of daughter buds of Mlc1-YFP-expressing cells was quantified in cultures growing either as yeast (B) or pseudohyphae (C) and plotted against bud length. In addition, the presence or absence of Mlc1-YFP fluorescence at the tip was recorded. The switch to isotropic growth occurs at a longer bud length and greater axial ratio in pseudohyphae compared to yeast. Note differences in scales. (D) Mlc1-YFP-expressing cells were induced to form hyphal germ tubes. After 105 minutes, cells were fixed and stained with DAPI and examined with a Delta Vision microscope. Mlc1-YFP fluorescence is visible as a spot at the tip and a ring at the site of septation in the germ tube. Nuclei stained with DAPI are visible as circular areas within the mother cell, or migrating into the germ tube, or dividing across the cytokinetic ring. (E) Hyphal germ tube showing Mlc1-YFP simultaneously at the tip and the septation ring. Scale bar, 5 µm (A,E); 15 µm (D).

View larger version (38K): [in a new window]   Fig. 10. Quantification of Mlc1 fluorescence in hyphae and pseudohyphae. Strains expressing either Mlc1-YFP or Spa2-YFP were cultured under the indicated conditions and the average peak fluorescence intensity was determined. An example of the 3D surface contour map of fluorescence is shown alongside the hyphal (A,D) or pseudohyphal (B) tip from which it was derived. (A) A ridge of lesser fluorescence surrounds a focused point of high fluorescence. (B) The contour map reveals only a structure whose maximum fluorescence resembles that of the ridge detected in A and no focal point of fluorescence. The histograms in A and B show the mean maximum fluorescence intensity in cells divided into bins according to bud length. Each bin consists of at least 100 cells. The time course experiments described in panels A and B have been carried out on three separate occasions with similar results. (C) Average peak fluorescence was compared between Spa2-YFP (black) and Mlc1-YFP (grey) in the different morphologic forms. Spa2 shows little variation in its peak intensity between growth forms whereas Mlc1 fluorescence is more than four times greater in hyphae (H) than pseudohyphae (PH) or yeast (Y). (D) The shape of the Spa2 fluorescence surface contour map in hyphae resembles that of Mlc1 in pseudohyphae shown in Panel B. Bar, 1 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter