Ordered kinetochore assembly in the human-pathogenic basidiomycetous yeast Cryptococcus neoformans.

UNLABELLED: Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE: Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

Introduction

High-fidelity chromosome segregation is essential for faithful propagation of genetic information. The process of chromosome segregation is powered by the dynamic interaction between the chromosomes and the spindle microtubules. The chromosomal attachment site of spindle microtubules is a multimeric proteinaceous structure formed on the centromere DNA, termed the kinetochore (KT) (1). An error in the KT-microtubule interaction can result in improper chromosome segregation, leading to aneuploidy, a hallmark of several cancers (2, 3).

While the trilaminar architecture of the KT is conserved from yeast to humans, structural and functional evolution of some of the KT proteins is evident (4). Among the three layers of the KT, the components of the inner layer that interact directly with DNA are conserved in organisms with regional centromeres (centromere DNA is >3 kb in length) (5). The centromere-specific histone H3 of the CENP-A/Cse4 family and CENP-C/Mif2 are two such evolutionarily conserved inner KT proteins (6). Several middle KT proteins, such as Mis12/Mtw1 and Nuf2, are present in most eukaryotes. In contrast, the 10-subunit outer KT Dam1 protein complex is found only in fungi (7–9).

Though the overall architecture of the KT is largely conserved, its assembly and regulation vary among species. Most metazoans undergo open mitosis. Ordered KT assembly is orchestrated with disassembly of the nuclear envelope (NE) in open mitosis, where the middle and outer KT assemble on the constitutive inner KT to allow access of microtubules to the centromere (10–12). Current knowledge about the dynamics of the fungal KT is primarily based on studies involving three model ascomycete species (6), the budding yeasts Saccharomyces cerevisiae and Candida albicans and the fission yeast Schizosaccharomyces pombe. Both budding yeasts undergo closed mitosis, as the NE never breaks down (13, 14) and the KT is fully assembled and attached to microtubules throughout the cell cycle (15). In contrast, in the fission yeast S. pombe, most proteins of the outer KT assemble only during mitosis, although the NE remains intact (16, 17).

Cryptococcus neoformans is a human pathogen known to cause meningitis primarily in immunocompromised but also in immunocompetent patients (18). C. neoformans has an 18.9-Mb genome distributed in 14 nuclear chromosomes and one 21-kb mitochondrial chromosome (19). By localizing several KT proteins, microtubules, and NE markers, we demonstrate, for the first time, that a metazoanlike ordered process of KT assembly exists in a fungal species. Our results suggest that mitotic events associated with metazoans—ordered KT assembly, interaction between KTs and microtubules only during mitosis, and opening of the NE—have evolved in fungal species.

RESULTS AND DISCUSSION

To study the dynamics of KT assembly in C. neoformans, we identified and fluorescently tagged proteins with homology to eukaryotic KT subunits that represent the three functional layers (Fig. 1A; see Table S1 in the supplemental material), as follows: CENP-A/Cse4 and CENP-C/Mif2 (inner KT components), Mis12/Mtw1 and Nuf2 (middle KT proteins), and Dad1 and Dad2, two constituents of the Dam1 complex (an outer KT-protein complex). Strains expressing the tagged proteins showed no obvious growth defects. In cells undergoing mitosis, each of these six fluorescently tagged proteins exhibited localization typical of a budding yeast KT protein, suggesting that each of these six putative KT protein homologs in C. neoformans is indeed localized at the KT (Fig. 1B).

FIG 1 

Nuclear division and dynamics of centromeres during cell cycle in C. neoformans. (A) Conserved proteins representing three layers of a fungal kinetochore that were chosen for the analysis. (B) In cells undergoing mitosis, putative kinetochore proteins tagged with mCherry (Cse4 [strain CNV101], Mif2 [strain CNV102], and Mtw1 [strain CNV103]) or GFP (Nuf2 [strain SHR515], Dad1 [strain CNV104], and Dad2 [strain SHR107]) show localization similar to that of the kinetochore in ascomycetous budding yeasts. (C) Cells expressing a chromatin marker, GFP-histone H4 (strain CNV108), were analyzed by time-lapse microscopy. During mitosis, the chromatin first moves entirely to the daughter cell and then condenses and forms a doubletlike structure (arrow). Finally, the chromatin divides into two compact signals between the daughter and mother cell. (D) Cells expressing GFP-tubulin and the inner kinetochore marker mCherry-Cse4 (strain LK275) were studied by time-lapse Z-section microscopy, and single focal planes are shown. Initially nonclustered mCherry-Cse4 converges into a single dot in the mother cell. In the daughter cell, mCherry-Cse4 rearranges into a doublet that surrounds a single rod of GFP-tubulin, a putative spindle (t = 40, arrow). At this stage, cytoplasmic GFP-tubulin cables are no longer visible. After mCherry-Cse4 divides between the daughter and mother cells, mCherry-Cse4 declusters and the GFP-tubulin cytoplasmic cables reappear (t = 90). See also Fig. S1 in the supplemental material. Bars, 5 µm.

C. neoformans centromeres are not clustered in nondividing cells.

In C. neoformans, similar to other basidiomycetous yeasts, nuclear division occurs within the daughter cell (20–23). To visualize the dynamics and changes associated with chromatin, we followed the localization of green fluorescent protein-tagged histone H4 (GFP-H4) (Fig. 1C). Consistent with previous reports, GFP-H4 transitioned entirely into the daughter cell to undergo division. In the daughter cell, the surface area of the GFP-H4 signal was reduced by ~66% compared to the signal before the nuclear mass migrated through the mother bud neck, suggesting significant chromatin condensation during mitosis. Notably, at this stage, GFP-H4 appeared as a doublet (Fig. 1C, arrow). Subsequently, the nuclear division occurred in the daughter cell; half of the nuclear mass migrated back into the mother cell, and the other half remained in the daughter cell.

We next examined the dynamics of KTs by performing time-lapse imaging of cells expressing fluorescently tagged CENP-A, mCherry-Cse4. To better discern stages of the cell cycle, the spindle was visualized with GFP-tagged α-tubulin (GFP-tubulin). In striking contrast to hemiascomycetous budding yeasts (S. cerevisiae and C. albicans), in which KTs are always clustered (9, 14, 24–27), mCherry-Cse4 showed multiple distinct dots in nondividing cells, including unbudded cells and cells with small buds (budding index less than or equal to ~0.4) (Fig. 1D; see Fig. S1A and B in the supplemental material). KTs are not usually clustered in most metazoans. Semiquantitative analysis of fluorescence intensity revealed that the number of mCherry-Cse4 dots was consistent with the number of centromeres (14 chromosomes per haploid cell) in C. neoformans (Fig. S1A). The circular arrangement of mCherry-Cse4 dotlike signals in most nondividing cells suggested localization adjacent to the nuclear periphery. Peripheral localization was further confirmed in cells that also expressed GFP-H4 or the NE marker protein GFP-Ndc1 (Fig. S1D and E). The number of dotlike signals of mCherry-Cse4 gradually decreased until, ultimately, in large budded cells (budding index of 0.4 to 0.55), the mCherry-Cse4 was visible as a single bright dot that subsequently migrated into the mother-bud neck (Fig. 1D; Fig. S1B). Interestingly, when clustering was nearly complete, mCherry-Cse4 appeared to contact a bundle of GFP-tubulin cables, suggesting that clustering of KTs and the attachment of microtubules to the KT occur concomitantly (Fig. 1D; Fig. S1A and B). Clustered mCherry-Cse4 was always at the NE, similar to hemiascomycetous budding yeasts (14, 24, 28, 29). In the daughter cell, a dotlike signal of GFP-tubulin underwent a transition into an elongated rod which was surrounded by 2 bars of mCherry-Cse4 when observed in a single focal plane (Fig. 1D, arrow; Fig. S1C). While the architecture of this microtubule KT arrangement remains to be determined, it may be functionally analogous to the metaphase plate of metazoans. At this stage, cytoplasmic microtubules were no longer visible. Within a minute or less after astral microtubules became visible, the mCherry-Cse4 signals divided into two separate dots, with one migrating back to the mother cell. After division of mCherry-Cse4, cytoplasmic microtubules reappeared and the mCherry-Cse4 began to decluster, forming dotlike signals similar to those observed in nondividing cells.

Microtubules are necessary for clustering of kinetochores prior to mitosis.

The concomitant localization of KTs and tubulin when KTs were clustered prompted us to examine whether microtubules play a role in the clustering of KTs. To test this, we used nocodazole (Noc) to depolymerize microtubules in a strain that coexpressed mCherry-Cse4 (to assess the position of centromeres) and GFP-Ndc1 (to assess the state of the NE and the stage during the cell cycle) (Fig. 2A). First, we obtained a population of mostly unbudded cells (96% unbudded) by limiting oxygen during growth (30). In this starting population, mCherry-Cse4 was not clustered (Fig. 2A). Approximately 70 min after release from the arrest, both the control and Noc-treated cells were mostly budded, indicating resumption of synchronized growth and confirming that Noc treatment did not affect the establishment of cell polarity and the subsequent growth of the bud. While 47% of the control cells had the mCherry-Cse4 signal either in the daughter cell or divided between the mother and the daughter, no such cells were found in the Noc-treated sample at 70 min (Fig. 2B). As expected, a significant portion of the control cells (21%) showed clustered mCherry-Cse4. The average ratio of the bud/mother size in these cells was 0.6 (standard deviation [SD] = 0.09). Based on this information, we examined Noc-treated cells with a bud/mother size ratio of ≥0.62 for clustering of mCherry-Cse4. Strikingly, we found that 39% of all Noc-treated cells had an average bud/mother size ratio of 0.72 (SD = 0.09) and nonclustered mCherry-Cse4 in the mother with no signal in the daughter. No Noc-treated cells with clustered mCherry-Cse4 signals were found at 70 min except for a small percentage (9%) of cells with aberrant localization of GFP-Ndc1. In these cells, chromatin was significantly compacted (data not shown) and GFP-Ndc1 formed a tight ring surrounding a small cluster of mCherry-Cse4 and/or formed a bright cluster away from mCherry-Cse4 (Fig. 2A). We hypothesize that, in these cells, a process of chromatin condensation bypassed the requirement for microtubules without hindering the formation of the trilayered KT structure and resulted in bringing centromeres in close proximity. In summary, our findings suggest that microtubules are involved in the clustering of centromeres prior to mitosis in C. neoformans. Clustering of KTs in S. cerevisiae during mitosis depends on the kinesin-8 homologue Kip3, while during interphase, it requires both microtubules and the KT-associated protein Slk19 (31, 32). Since the sequence homolog of Kip3 in C. neoformans remains uncharacterized and there is no obvious homologue of Slk19, the precise contribution of microtubules to clustering of centromeres will require further studies.

FIG 2 

Clustering of centromeres requires microtubules. (A) Cells expressing mCherry-Cse4 and GFP-Ndc1 (strain CNV111) were synchronized in G1/G2 and released from the arrest in the presence of nocodazole (1 µg/ml) or dimethyl sulfoxide (DMSO) as a control. At 70 min after the release, the majority of nocodazole-treated cells showed nonclustered mCherry-Cse4, while in the control sample, the majority of cells either showed clustered centromeres or progressed through mitosis. (B) Quantification of cells with respect to the stage of the cell cycle and percentage of centromere clustering. Bar, 5 µm.

Kinetochores in C. neoformans assemble in an ordered manner.

The appearance of nonclustered centromeres in C. neoformans suggests a fundamental difference in centromere dynamics between C. neoformans and the ascomycetous budding yeasts where such studies have already been performed. This led us to analyze the localization dynamics of the KT proteins from all 3 KT layers in C. neoformans. An inner KT protein, Mif2-mCherry, showed persistent localization similar to that of GFP-Cse4, suggesting that the inner KT is constitutively present at the centromere (Fig. 3A and B). In contrast, the middle KT protein Mis12/Mtw1 and the outer KT protein Dad1 did not colocalize with inner KT proteins Cse4 and Mif2 in cells that showed nonclustered KTs (Fig. 3C and D). Thus, the middle and outer KT do not assemble at the stage when KTs are nonclustered. This possibility was further confirmed by using another set of proteins, the middle KT protein GFP-Nuf2 and an outer KT component, GFP-Dad2 (see Fig. S2A and B in the supplemental material). Based on these results, we conclude that the middle and outer KT layers form only during mitosis. The relative localization timing of the GFP-Dad1 and the Mtw1-mCherry in the same cells revealed that the GFP-Dad1 signals became visible later than the appearance of Mtw1-mCherry but disappeared before Mtw1-mCherry (Fig. 3E). An analysis of the budding index of the cells examined (n = 100) confirmed a difference in the timing of the appearance of middle KT (budding index equal to 0.4) and outer KT proteins (budding index equal to 0.55; P = 0.0001) (Fig. S2C). Taken together, these results indicate that the assembly of the KT in C. neoformans is an ordered process in which the inner KT remains assembled on the centromere DNA throughout the entire cell cycle and components of the middle KT are incorporated prior to mitosis, concomitant with KT clustering, followed by complete assembly of the KT just before KTs start moving to the daughter cell (Fig. 3F). This process has not been described in yeasts and is strikingly similar to the dynamics of KT assembly in metazoans. Thus, our study suggests evolutionary conservation of KT assembly from basidiomycete yeasts to humans.

FIG 3 

Ordered kinetochore assembly in C. neoformans. (A) Two inner kinetochore proteins, GFP-Cse4 and Mif2-mCherry (strain CNV115), colocalized at all stages of the cell cycle. Both proteins colocalized as separate dots in unbudded cells (first column), small-budded cells (not shown) and soon after cytokinesis (last column) but remained clustered during mitosis (middle three columns). (B) A 3-dimensional (3-D) reconstruction based on Z-stack images of an unbudded cell shows a complete overlap of GFP-Cse4 and Mif2-mCherry. (C) The middle kinetochore protein Mtw1-mCherry was not visible in cells where the inner kinetochore protein GFP-Cse4 was found as multiple nonclustered signals (strain CNV116; the first and the last columns). However, Mtw1-mCherry colocalized with clustered GFP-Cse4 (middle three columns). (D) The outer kinetochore component GFP-Dad1 became visible prior to mitosis when it colocalized with the inner kinetochore protein Mif2-mCherry in a single cluster (strain CNV118). (E) Colocalization of an outer (GFP-Dad1) and a middle (Mtw1-mCherry) kinetochore protein showed that GFP-Dad1 was loaded onto the kinetochore later than Mtw1-mCherry (strain CNV117). GFP-Dad1 was visible during mitosis (t = 16 to 27 min) and disappeared soon after chromosome segregation, while Mtw1-mCherry remained present (t = 32 min). (F) Schematic showing ordered assembly of kinetochore proteins. The inner, middle, and outer kinetochore protein names are highlighted in blue, yellow, and green, respectively. See also Fig. S2 in the supplemental material. Bars, 5 µm (A, C, D, and E) and 1 µm (B).

The nuclear envelope opens partially during mitosis in C. neoformans.

Ordered KT assembly and open mitosis where the NE breaks down are hallmarks of metazoan mitosis. Having established metazoanlike ordered assembly of the KT in C. neoformans, we sought to examine the status of the NE in this fungal pathogen. First, we followed the localization of the integral NE protein Ndc1 (33, 34). GFP-Ndc1 outlined the NE throughout the entire duration of nuclear division (Fig. 4A). Approximately half of the GFP-Ndc1 signal that outlined the NE remained in the mother cell, while clustered mCherry-Cse4 led the rest of the GFP-Ndc1-marked NE into the daughter cell at the onset of mitosis. In a closer look, the GFP-Ndc1 fluorescent signal immediately adjacent to the clustered mCherry-Cse4 appeared somewhat weaker or discontinuous (Fig. 4B). These data suggest that the NE remains largely intact during mitosis in C. neoformans and only a partial opening of the NE takes place, specifically, near clustered KTs. Further analysis of the NE using transmission electron microscopy (TEM) revealed that, while the NE stayed largely intact during mitosis in C. neoformans, it did rupture at the point where the spindle broke through the NE during the migration of the spindle into the daughter cell (Fig. 4C; see Table S2 in the supplemental material). TEM indicated that the nucleolus was not present during mitosis (Table S2). This was confirmed with the strain expressing the nucleolar marker GFP-Nop1 (Fig. 4D, t = 20 min). We also followed the localization of the fluorescently tagged Nup107, an essential component of the nuclear pore complex (NPC) (35). Time lapse imaging of GFP-Nup107 and mCherry-Cse4 showed that the GFP-Nup107 signal disappeared during the stage when mCherry-Cse4 was present in the daughter cell (Fig. 4E). Based on these results, we propose that C. neoformans undergoes a semiopen mitosis characterized by a partial breakage of the NE near spindle pole bodies (SPBs) and complete disassembly of the NPC (Fig. 5A). Recently, various types of mitosis other than the conventional open or closed mitosis have been reported in fungi. A semiopen type of mitosis was described in Aspergillus nidulans, where some of the NPC proteins disassemble (36). Another type of mitosis was reported in the fission yeast Schizosaccharomyces japonicus, where the NE breaks during anaphase (37, 38). In Ustilago maydis, another basidiomycetous yeast, the NE breaks near SPBs and NPCs disassemble completely (22, 39). The type of mitosis in C. neoformans appears similar to that of U. maydis, with subtle differences in the dynamics of the NE. In U. maydis, the NE remains in the mother cell and does not enclose chromatin during mitosis, but in C. neoformans, a part of the NE moves to the daughter cell and encloses the DNA during division.

FIG 4 

The nuclear envelope in C. neoformans breaks open partially during mitosis. (A) The nuclear membrane (visualized by GFP-Ndc1 in strain CNV111) was largely intact throughout the cell cycle. Clustered mCherry-Cse4 led an invagination of the nuclear membrane into the daughter cell at the onset of mitosis (t = 2 to 3 min) (B) A 3-D reconstruction of Z-stack images of a cell from panel A at t = 0 showed a discontinuous signal of GFP-Ndc1 at the site where mCherry-Cse4 was clustered, suggesting partial opening of the nuclear membrane near clustered kinetochores. Panels 4, 5, and 6 show projections of the area in panel 2 indicated with the arrow. The red line in panel 5 indicates the surface of the medium. (C) TEM analysis of mitosis in C. neoformans. In images 2 to 6, the daughter cell is the one on top (smaller than the mother cell). Images: 1 and 7, G1-S phase; 2 and 8, G2 phase; 3 and 9, prophase; 4, 10, and 11, prometaphase; 5 and 12, metaphase; 13, anaphase; 6, 14, and 15, telophase. Nuclear envelope (NE) was intact at G1 through prophase, had broken near the spindle pole body (arrow) at prometaphase, and was closed at the end of telophase. Nucleolus (NU) was visible at G1 through prophase, stayed in the mother cell at prometaphase, disappeared at metaphase, and reappeared after telophase. Spindle pole body resided on the nuclear envelope as one duplicated form at G1 though G2 phase, separated into two at prophase, entered the nuclear region by breaking the nuclear envelope at prometaphase, was located at the spindle poles at metaphase and anaphase, and was extruded back to the cytoplasm from the nuclear region at telophase. Microtubules (arrowheads) were distributed in the cytoplasm at G1 through prophase and appeared in the nucleus (N) at prometaphase through middle of telophase. (D) Time lapse imaging with the nucleolus marker GFP-Nop1 showed that the nucleolus disappeared during mitosis when mCherry-Cse4 was present in the doublet stage (strain LK353). (E) Time-lapse analysis of nuclear pore protein GFP-Nup107 and mCherry-Cse4 (strain LK317). GFP-Nup107 was not present on the NE during metaphase (t = 9 to 11.15 min), suggesting that NPCs disassembled at this stage. After genomic division, GFP-Nup107 was again visible on the NE, marking the reassembly of NPCs after mitosis. Bars, 5 µm (A, B [top], D, and E), 1 µm (B [bottom] and C [1 to 6]), 250 nm (C [7 to 15]).

FIG 5 

Key events during mitotic cell cycle in C. neoformans. (A) A model showing clustering of centromeres, a gradual change in kinetochore architecture, and the nuclear envelope dynamics during the progression of the cell cycle in C. neoformans. The budding index at each stage was calculated by measuring the relative bud sizes of at least 100 cells. (B) A comparison of key mitotic events in S. cerevisiae, C. neoformans, and humans.

Taken together, the analysis of nuclear division in C. neoformans suggests that, in basidiomycetous budding yeasts, KTs assemble in an ordered manner in concert with partial disassembly of the NE (Fig. 5A). We propose that this form of mitosis was an ancestral mode present in basidiomycetes and the mitotic events in hemiascomycetous budding yeasts might have evolved independently (Fig. 5B). The intriguing nonclustered state of centromeres and the apparent localization near the NE in nondividing cells are reminiscent of the localization reported in some metazoan cells (40–43). As clustering of centromeres in C. neoformans appears to be completed before assembly of the outer KT, microtubule-mediated clustering of centromeres may not occur simply via the attachment of microtubules to the assembled KT. Further studies on mechanisms of KT clustering and their assembly in C. neoformans should lead to a better understanding of the evolution of mitosis in eukaryotes.

MATERIALS AND METHODS

Strains and plasmids.

The strains and plasmids used in this study are listed in Table S3 in the supplemental material. C. neoformans culture was grown in yeast extract-peptone-dextrose (YPD) medium at 30°C.

Construction of fluorescently tagged proteins.

Fluorescent fusion proteins were made by either cloning or overlap PCR. The list of primers used for construction is given in Table S4 in the supplemental material. Plasmids or overlap PCR products to express GFP and mCherry fusion proteins were mainly constructed using a method described previously (44) (See Text S1 in the supplemental material for detail).

Two rounds of transformation were used to tag the two different proteins in the same strain, and medium containing both nourseothricin (NAT) and G418 was used for selection. The transformants were screened by fluorescence microscopy.

Fluorescence microscopy.

Cells were grown in the YPD medium for 14 to 16 h and pelleted at 4,000 rpm. The cells were then washed once with distilled water and finally resuspended in distilled water. The cells were placed on slides, covered with coverslips, and observed under a microscope. For live-cell imaging, an overnight YPD or synthetic complete medium-grown culture was diluted in fresh synthetic complete growth medium and grown for 3 h. Next, ~0.5 µl of cell suspension was placed on a slide containing a thin complete growth medium–2% agarose patch and covered with a coverslip. Images were captured at 100× using either a Carl Zeiss confocal laser scanning microscope (LSM 510 META) or the DeltaVision system (Olympus IX-71 base) equipped with a CoolSnap HQ2 high-resolution charge-coupled-device (CCD) camera and a 100× objective (100×/1.40 oil UPLSAPO100X0 1-U2B836 WD 120-µm differential interference contrast [DIC] ∞/0.17/FN26.5, UIS2 series). The filters used (Chroma) were GFP/fluorescein isothiocyanate (FITC) 475/28, mCherry/AF594 575/25 for excitation and GFP/FITC 525/50, mCherry/AF594 632/60 for emission and a Dual em pass GFP/mCherry filter for high-speed imaging. For more detailed description of the DeltaVision system, see http://microscopy.duke.edu/dv.html. For confocal microscopy, Ar 488 and HeNe 543 lasers were used for excitation of GFP and mCherry, respectively. The image processing was done using the Zeiss image-processing software LSM 5 Image Examiner or ImageJ, Image-Pro Plus, or Photoshop (Adobe Systems, San Jose, CA).

Supplemental methods. Download

Text S1, PDF file, 0.4 MB

Localization and dynamics of centromeres. (a) Time-lapse Z-section analysis of cells expressing mCherry-Cse4 and GFP-tubulin (strain LK275). Distribution of mCherry-Cse4 clusters with indicated number of centromeres in each cluster (number in an oval) is shown. Each oval represents a class of a cluster characterized by the number of associated centromeres. For instance, cell 1 in the first time point has 11 clusters containing one centromere, 4 clusters containing 2 centromeres, and 3 clusters containing 3 centromeres (a total of 18 clusters). Numbers were estimated based on measurements of the fluorescence intensity of each signal following 3-D reconstruction. In small budded cells, centromeres were declustered (top panel). With progression of the cell cycle, centromeres clustered gradually to form a single distinct dot in cell 1 and two clusters in cell 2. In the last time point shown for cell 2, only one cluster (containing 22 centromeres) is visible in the view of a single focal plane. A 3-D reconstruction of this cell revealed that a second cluster, containing an estimated 6 centromeres, lagged behind and was not in contact with the GFP-tubulin cables. A later time point showed that all centromeres in cell 2 clustered eventually. (b) A 3-D reconstruction of a cell 2 from A showing a change from declustered to mostly clustered mCherry-Cse4 contacting a net of microtubule cables. (c) A 3-D reconstruction and a model showing the mCherry-Cse4 and GFP-tubulin arrangement formed in the daughter cell during metaphase. (d) Imaging of cells expressing mCherry-Cse4 and GFP-H4 (strain CNV114) showed localization of centromeres with respect to the nuclear mass. Centromeres were present on the periphery of the nucleus throughout the cell cycle. In the declustered state, the centromeres formed a ringlike structure around the nuclear mass (first column). The clustered centromeres, at later stages of the cell cycle, were also localized to the periphery of the nuclear mass (columns 2 and 3). Subsequently, these clustered centromeres were found to be localized between the nuclear mass (column 4). Finally, during division, the kinetochore cluster came to the periphery of the nuclear mass again (column 5 and 6). (e) A 3-D reconstruction of cells showing that mCherry-Cse4 is present near the NE marked by GFP-Ndc1 (strain CNV111). Bars, 1 µm (B, C, and E) and 5 µm (A and D). Download

Figure S1, PDF file, 0.6 MB

Localization of middle and outer kinetochore proteins. (a) Two middle kinetochore proteins, Mtw1-mCherry and GFP-Nuf2, colocalized completely during mitosis and were not visible at other stages of the cell cycle (strain SHR516). (b) Two outer kinetochore proteins, GFP-Dad1 and Dad2-mCherry, colocalized and showed a pattern of localization similar to that of the middle kinetochore proteins (strain CNV119). (c) An outer kinetochore component, GFP-Dad1, assembles at the kinetochore later than the middle kinetochore protein Mtw1-mCherry (strain CNV117). Budding index was calculated for cells expressing either GFP-Dad1, Mtw1-mCherry, or mCherry-Cse4 (n = 100). In cells with clustered centromeres before mitosis, the budding index was significantly higher for GFP-Dad1 than for Mtw1-mCherry (P = 0.0001). Bars, 5 µm. Download

Figure S2, PDF file, 0.5 MB

Kinetochore proteins in Cryptococcus neoformans.

Table S1, PDF file, 0.1 MB.

TEM analysis at various cell cycle stages.

Table S2, PDF file, 0.1 MB.

Strains and plasmids used.

Table S3, PDF file, 0.4 MB.

Primers used in this study.

Table S4, PDF file, 0.1 MB.