ABSTRACT
Candida species cause severe infections like invasive candidiasis, which annually affects 1.5 million people worldwide and causes close to 1 million deaths. Candida albicans is the predominant cause of candidiasis. We previously showed that Eps15-Homology domain-containing protein Irs4p binds 5-phosphatase enzyme Inp51p to regulate plasma membrane levels of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) in C. albicans. Indeed, deletion of IRS4 or INP51 led to elevated levels of PI(4,5)P2 and the presence of abnormal intracellular membranous PI(4,5)P2 patches. We demonstrated an interplay between PI(4,5)P2 and septins to regulate the PKC-Mkc1 cell wall integrity pathway, echinocandin and cell wall stress responses, and virulence during candidiasis. To gain insights into the nature of these abnormal patches, we used fluorescent protein tagging and live-cell imaging to follow their nascency. We show that these abnormal patches tightly correlate with cytokinesis, as they predominantly arise close to the site and time of cell division. We further demonstrate that these patches colocalize PI(4,5)P2 with actomyosin ring components Act1p and Myo1p, which form its core, and active Rho1p, a small GTPase that plays a regulatory role. Additionally, activation of Rho1p was altered in irs4 and inp51 mutants compared to wild-type strain, with over-activation or down-activation during early exponential or stationary phase, respectively. Wild-type cells exposed to 4× MIC of the echinocandin caspofungin show abnormal PI(4,5)P2 patches that colocalize with the same cytokinesis components as above, except that they are transient. Taken together, our results support a model in which PI(4,5)P2 plays a pivotal role, along with Rho1p, in the correct execution of cytokinesis and response to caspofungin.
KEYWORDS: phosphatidylinositol-(4,5)-bisphosphate; Candida albicans; cytokinesis; echinocandin; Rho GTPase RHO1; actin; myosin; caspofungin; INP51; IRS4; Yeast
INTRODUCTION
Candida infections are caused by diverse commensal, opportunistic budding yeast species from distinct families in the Saccharomycetales order (1). These infections manifest different clinical forms ranging from non-life-threatening superficial mucocutaneous infections like oropharyngeal and vulvovaginal candidiasis, or superficial skin infections, to the potentially lethal deep-seated and disseminated invasive candidiasis (IC). Mortality rates from IC can reach 20%–40% for immunocompromised individuals and hospitalized patients with serious underlying diseases, and they pose a significant economic burden to healthcare systems (2). Candida albicans is the leading cause of IC. Echinocandins are the agents of choice against most infections by C. albicans and other Candida spp. These drugs exert fungicidal activity on Candida by damaging the cell wall through inhibition of 1,3-β-D-glucan synthase, a critical enzyme in cell wall biogenesis.
The Candida cell wall and adjoining plasma membrane (PM) are crucial to the pathogenesis of IC and echinocandin responsiveness (3). Phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) localizes to the PM’s cytoplasmic layer, where it anchors via its fatty acid chain and glycerol backbone (4). This leaves its inositol headgroup available to interact conformationally with PH domain proteins (5) or electrostatically with basic residues of arginine and lysine (6). In previous work, we found that exposure of wild-type C. albicans cells to the echinocandin caspofungin led to an elevation of PI(4,5)P2 levels in a rapid and dose-dependent fashion and a dynamic mislocalization of both PI(4,5)P2 and septins within aberrant PM invaginations (7). Many C. albicans cells exposed to caspofungin also demonstrated abnormally wide bud necks (8). In other studies, we showed that the C. albicans Eps15-Homology domain-containing protein Irs4p binds the 5-phosphatase enzyme Inp51p to regulate intracellular levels of PI(4,5)P2. C. albicans mutant strains with deletion of genes encoding either Irs4p or Inp51p over-activated the PKC-Mkc1 cell wall integrity pathway upon caspofungin exposure, and they were hyper-susceptible to the drug and attenuated for virulence in a hematogenous disseminated mouse model of IC (9, 10). Mutant strains had elevated levels of PI(4,5)P2 and exhibited PI(4,5)P2-containing PM invaginations that resembled those of wild-type C. albicans exposed to caspofungin. These abnormal invaginations also encompassed septins (Sep7p and Cdc10p), chitin, glycosylphosphatidylinositol-anchored proteins (Rbt5p), and cell wall material (7, 9, 10). Taken together, our previous data indicate that PI(4,5)P2-septin regulation is crucial for echinocandin response, cell wall integrity, and C. albicans virulence. The data, including mislocalization of PI(4,5)P2 and septins in abnormal patches (7) and abnormally wide bud necks in wild-type cells exposed to caspofungin (8), point to a possible link between PI(4,5)P2 and cytokinesis.
While many research investigations established the involvement of PI(4,5)P2 or its degradation, in several functions like cytoskeleton remodeling, endocytosis, exocytosis, ion channel regulation, and signaling pathways (4, 11), only a handful of studies provided evidence hinting to the possible role of PI(4,5)P2 in cytokinesis (12). In the current study, we provide the first strong evidence implicating PI(4,5)P2 in cytokinesis in C. albicans. We further show its importance for echinocandin responses. Using live-cell imaging, we first looked at the spatio-temporal connection between the appearance of PI(4,5)P2 patches and cytokinesis in irs4 and inp51 mutant cells. Then, we examined colocalization of PI(4,5)P2 in the mutants with the main actors of cytokinesis machinery: the major components of the actomyosin ring (Act1p and Myo1p) and the GTP-binding protein Rho1p, which is an upstream activator of the PKC-Mkc1 cell wall integrity pathway, a regulator of the actin cytoskeleton during cytokinesis, and a regulatory subunit of 1,3-β-D-glucan synthase. Finally, we determined if some of the abnormalities in mutant cells were also evident in wild-type C. albicans cells exposed to caspofungin, in which PI(4,5)P2 levels are increased.
MATERIALS AND METHODS
Strains, growth conditions, PCR, and subcloning
C. albicans strains used in this study are listed in Table 1. Strains were grown in yeast-peptone-dextrose (YPD) or synthetic defined (SD) media at 30°C. All oligonucleotides used in this study are available upon request. PCR was performed using high-fidelity polymerases, including Agilent’s pfuUltra II Fusion HS DNA polymerase (Santa Clara, CA, USA) or, for long PCR fragments (5–12 kb), Takara’s PrimeSTAR Max DNA polymerase (Mountain View, CA, USA). For on-colony PCR screening, we used 5PRIME HotMaster Taq DNA Polymerase (Quantabio, Beverly, MA, USA). PCR conditions followed the manufacturer’s recommendations; the annealing temperature ranged from 54°C to 58°C, and the elongation time was 15–30 s/kb. To perform chimeric protein fusion, PCR bands were purified with Nucleospin Gel and PCR Clean-up Kit (Takara BIO USA) and ligated with Quick Ligase Kit (New England Biolabs, Ipswich, MA, USA). The ligated PCR fragment ends should have corresponding oligos with a 5′ phosphorylation. Nested PCR was performed to amplify the fused open reading frames (ORF), using primers carrying appropriate restriction enzyme sites for subcloning into pSFS2A plasmid (13). We previously integrated into this plasmid: CaADH1 promoter and terminator to control the expression of the fused ORFs; F1 and F2 DNA fragments for integration by recombination into a noncoding genomic region between coordinates 625,000 and 627,000 of chromosome 1, upstream of the RP10 locus; and either CaGFP or CaRFP for fluorescent tagging (7, 8). NEB 10-beta chemically competent E. coli were used for plasmid transformation (New England Biolabs, Ipswich, MA, USA), which was carried out following the manufacturer’s heat-shock protocol. This E. coli strain was particularly efficient in transforming large DNA (10–14 kb).
TABLE 1.
List of strains used in this study
| Strain | Parent | Genotype and description | Research purpose | Reference |
|---|---|---|---|---|
| SC5314 | Clinical isolate | Wild type | (14) | |
| irs4_2KO | SC5314 | irs4Δ/irs4Δ | irs4 null mutant | (9) |
| inp51_2KO | SC5314 | inp51Δ/inp51Δ | inp51 null mutant | (10) |
| SC_AGPH | SC5314 | C1_625–7k::CaPHx2-GFP | PH-GFP expression | (8) |
| irs4_AGPH | irs4_2KO | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-GFP | PH-GFP expression | (8) |
| inp51_AGPH | inp51_2KO | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-GFP | PH-GFP expression | (8) |
| SC_AGPH-RC10 | SC_AGPH | C1_625–7k::CaPHx2-GFP/C1_625–7k::CDC10-RFP | PH-GFP and CDC10-RFP co-expression | (8) |
| irs4_ AGPH-RC10 | irs4_AGPH | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::CDC10-RFP | PH-GFP and CDC10-RFP co-expression | (8) |
| inp51_ AGPH-RC10 | inp51_AGPH | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::CDC10-RFP | PH-GFP and CDC10-RFP co-expression | (8) |
| SC_AGPH-RA1 | SC_AGPH | C1_625–7k::CaPHx2-GFP/C1_625–7k::ACT1-RFP | PH-GFP and ACT1-RFP co-expression | This study |
| irs4_ AGPH-RA1 | irs4_AGPH | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::ACT1-RFP | PH-GFP and ACT1-RFP co-expression | This study |
| inp51_ AGPH-RA1 | inp51_AGPH | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::ACT1-RFP | PH-GFP and ACT1-RFP co-expression | This study |
| SC_AGMy1 | SC5314 | C1_625–7k::MYO1-GFP | MYO1-GFP expression | This study |
| SC_AGPH-RMy1 | SC_AGPH | C1_625–7k::CaPHx2-GFP/C1_625–7k::MYO1-RFP | PH-GFP and MYO1-RFP co-expression | This study |
| irs4_ AGPH-RMy1 | irs4_AGPH | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::MYO1-RFP | PH-GFP and MYO1-RFP co-expression | This study |
| inp51_ AGPH-RMy1 | inp51_AGPH | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-GFP/C1_625–7k::MYO1-RFP | PH-GFP and MYO1-RFP co-expression | This study |
| SC_ARPH | SC5314 | C1_625–7k::CaPHx2-RFP | PH-RFP expression | This study |
| irs4_ARPH | irs4_2KO | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-RFP | PH-RFP expression | This study |
| inp51_ARPH | inp51_2KO | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-RFP | PH-RFP expression | This study |
| SC_ARPH-GP1RBD | SC_ARPH | C1_625–7k::CaPHx2-RFP/C1_625–7k::Pkc1-RBD-GFP | PH-RFP and Pkc1-RBD-GFP co-expression | This study |
| irs4_ARPH-GP1RBD | irs4_ARPH | irs4Δ/irs4Δ and C1_625–7k::CaPHx2-RFP/C1_625–7k::Pkc1-RBD-GFP | PH-RFP and Pkc1-RBD-GFP co-expression | This study |
| inp51_ARPH-GP1RBD | inp51_ARPH | inp51Δ/inp51Δ and C1_625–7k::CaPHx2-RFP/C1_625–7k::Pkc1-RBD-GFP | PH-RFP and Pkc1-RBD-GFP co-expression | This study |
| SC_RHOMYC | SC5314 | ROH1/RHO1-CMYC | Active-Rho1 pull-down and negative control | This study |
| irs4_RHOMYC | irs4_2KO | irs4Δ/irs4Δ and ROH1/RHO1-CMYC | Active-Rho1 pull-down | This study |
| inp51_RHOMYC | inp51_2KO | inp51Δ/inp51Δ and ROH1/RHO1-CMYC | Active-Rho1 pull-down | This study |
| SC_RHOMYC-GP1RBD | SC_RHOMYC | ROH1/RHO1-CMYC and C1_625–7k::Pkc1-RBD-GFP | Active-Rho1 pull-down | This study |
| irs4_RHOMYC-GP1RBD | irs4_RHOMYC | irs4Δ/irs4Δ, ROH1/RHO1-CMYC, and C1_625–7k::Pkc1-RBD-GFP | Active-Rho1 pull-down | This study |
| inp51_RHOMYC-GP1RBD | inp51_RHOMYC | inp51Δ/inp51Δ, ROH1/RHO1-CMYC, and C1_625–7k::Pkc1-RBD-GFP | Active-Rho1 pull-down | This study |
Transformation
DNA for transformation was prepared by digestion of plasmid followed by gel purification of the band of interest or by PCR amplifying the whole cassette (10–14 kb) using Takara’s PrimeSTAR Max DNA polymerase with M13 and T7 primers, then purifying the PCR band. Transformation of C. albicans was done using an electroporation protocol described previously (13). Transformants were selected on YPD plates supplemented with 200 µg/mL of nourseothricin (13). Positive transformants were screened by microscopy and PCR.
Live-cell imaging
C. albicans cells were grown overnight in YPD medium at 30°C. On the next day, cells were subcultured in fresh YPD at 30°C for 3–4 hours, and 150 µL of culture were deposited on a 35 mm glass-bottom dish (Matek, Ashland, MA). The dishes were pretreated with 10 µg/cm2 of Cell-Tak adhesive (BD Biosciences, Bedford, MA) per the manufacturer’s instructions to adsorb a thin layer of polyphenolic proteins on which cells were immobilized for microscopy. Dishes with the culture were incubated at 30°C for 1 hour. The medium was then removed, and the cells were washed twice with sterile double-distilled sterile water and once with fresh YPD to remove unattached cells. Finally, 150 µL of fresh YPD medium (or YPD supplemented with the appropriate amount of caspofungin) was added, and the dishes were placed in the heated stage of the microscope. Microscopy was performed at the University of Pittsburgh Center for Biologic Imaging, using established protocols (7, 8). We used a Nikon A1 confocal microscope for live-cell imaging and acquired data with NIS Elements software (Nikon, Minato, Tokyo, Japan).
Pulldown assay for active Rho1
Similar to the subcloning method described above, RHO1 was fused to a 13xcMyc tag, and a construct was generated to replace the native gene with the tagged version using the SAT flipper tool (13). After transforming cells with this cassette, transformants were screened with PCR to ensure correct integration. Furthermore, we used a western blot as described before (8) with anti-cMyc monoclonal antibody 9E10 (Thermo Fisher Scientific, Waltham, MA, USA) to confirm expression and correct size of the tagged protein. On the other side, the Rho1-binding domain from C. albicans PKC1 (PKC-RBD [15]) was amplified and fused to CaGFP and transformed for coexpression with Rho1p-13xcMyc. Cells were grown and harvested after overnight culture or after an additional 4 hours subculture. Proteins were extracted by cell disruption using a Mini-Beadbeater-8 (Biospec, Bartlesville, OK, USA) in NP-40 lysis buffer supplemented with protease inhibitors, including a Protease Inhibitor Cocktail and phenylmethylsulfonyl fluoride (Sigma-Aldrich, Saint-Louis, MO, USA). Active Rho1-13xMyc, interacting with PKC-RBD-CaGFP, was pulled down using the GFP-Trap Agarose kit (Chromotek, Planegg, Germany). Finally, a western blot with 9E10 anti-cMyc monoclonal antibody was used to detect active Rho1p. A strain expressing Rho1-13xMyc alone, without the PKC-RBD-CaGFP, was used with the GFP-Trap Agarose kit as a pulldown negative control.
RESULTS
PI(4,5)P2 patches correlate with cytokinesis in space and time
Mutant C. albicans inp51 and irs4 cells expressing the pleckstrin homology (PH) domain fused to CaGFP were used for live-cell imaging with confocal microscopy. We have previously shown that this PH domain, which is encoded by the human phospholipase C-δ1 (PLC), binds specifically in vivo to PI(4,5)P2 in C. albicans (7, 16, 17). Generally, about 50% of cells showed the PI(4,5)P2 patches. We followed these abnormal PI(4,5)P2 patches and scored their appearance with regard to proximity in space to the site of cell division or concurrence during cytokinesis. There was a tight association between abnormal PI(4,5)P2 patches and cytokinesis, as 83% and 84% of them appeared during and in proximity to cytokinesis, respectively (Table 2). Only 12% of them were not associated with either cytokinesis sites or timing. Representative examples of these events are shown in Fig. 1 and in time-lapse videos (Video S1a and b). These observations strongly link these abnormal PI(4,5)P2 patches with cytokinesis in time and space.
TABLE 2.
Scoring of PI(4,5)P2 patches with regard to cytokinesis spatio-temporal dynamicsa
| Cytokinesis site | Elsewhere | Total | |
|---|---|---|---|
| During Cytokinesis | 87 (78%) | 6 (5%) | 93 (83%) |
| Not during Cytokinesis | 7 (6%) | 12 (11%) | 19 (17%) |
| Total | 94 (84%) | 18 (16%) | 112 (100%) |
During 3-hour live cell imaging experiments, the appearance of the patches were scored to occure near the cytokinesis site or elsewhere, and during or not during cytokinesis. Shown in each case are the total number of occurences scored, and bold number between parenthesis are percentages.
Fig 1.
The abnormal PI(4,5)P2 patches are linked to cytokinesis in space and time. Three minutes interval time-lapse of dividing cells from inp51 mutant (similar results with irs4) expressing CaPHx2-CaGFP and CDC10-CaRFP, showing dynamics of the appearance of the abnormal PI(4,5)P2 patches, which are formed close to cytokinesis in space and time. White arrows depict two division sites in which the abnormal PI(4,5)P2 patches appear right after the end and close to the site of cytokinesis (see Video S1a and b).
Act1p and Myo1p colocalize with the abnormal PI(4,5)P2 patches
Actin and type II myosin heavy chains are encoded by ACT1 and MYO1, respectively, and are highly conserved across animal and yeast species. They are the main components of actomyosin, which forms the contractile ring that functions and is essential during cytokinesis (18). We transformed mutant cells expressing CaPHx2-CaGFP with a cassette to constitutively co-express CaACT1-CaRFP. Using a similar approach, we previously showed that septins (CaSep7p and CaCDC10p) colocalized with abnormal PI(4,5)P2 patches in mutant strains. In wild-type cells, Act1p was found localized to cortical actin patches, as expected. In mutants, however, Act1p was mislocalized and colocalized with the abnormal PI(4,5)P2 patches (Fig. 2; Video S2). Likewise, we co-expressed CaMYO1-CaRFP to localize myosin. In live-cell confocal imaging of wild-type cells, Myo1p initially localized to the bud neck, then translocated from the mother cell to the apical tip of the daughter cell during S-phase. At the end of anaphase and just before the completion of cell division, Myo1p migrated to the site of cell division as cytokinesis was completed (See Video S3a and b). In irs4 or inp51 mutant cells during cytokinesis, Myo1p is mislocalized and colocalized with abnormal PI(4,5)P2 patches as the patches emerge (Fig. 3; Video S3c). These data show the main components of actomyosin, an essential element of cytokinesis machinery, to be mislocalized with PI(4,5)P2 patches.
Fig 2.
Act1p colocalizes with the abnormal PI(4,5)P2 patches. Confocal microscopy of irs4 (or inp51) mutant expressing CaPHx2-CaGFP and ACT1-CaRFP. The left panel shows the GFP signal, the center panel shows a merging of the differential interference contrast (DIC) and RFP signal, and the right panel shows the merging of GFP and RFP signals. White arrows depict the co-mislocalization of PI(4,5)P2 and Act1p in the abnormal PI(4,5)P2 patches (see Video S2a and b).
Fig 3.
Myo1p colocalizes with the abnormal PI(4,5)P2 patches. Confocal microscopy of irs4 (or inp51) mutant expressing CaPHx2-CaGFP and CaMYO1-CaRFP. The left panel shows the GFP signal, the center panel shows a merging of the DIC and RFP signal, and the right panel shows merging of GFP and RFP signals. White arrows depict the co-mislocalization of PI(4,5)P2 and Myo1p in the abnormal patches (see Video S3c).
Active Rho1p is mislocalized to the abnormal PI(4,5)P2 patches, with altered activation in irs4 or inp51 mutant cells
In baker’s yeast, the small GTPase Rho1p, an essential protein, has been implicated in cell polarization, actin organization, and cell wall synthesis (19–21). One of the effectors of Rho1p is Pkc1, a protein kinase that signals to the Mpk1 MAP kinase cascade to control actin cytoskeleton organization and cell wall biosynthesis genes (22–25). Pkc1p was shown to specifically bind active GTP-bound Rho1p via the Rho1-binding domain (RBD) (22). As a reporter for active Rho1, we fused PCR-amplified C. albicans Pkc1p-RBD (aa 371–636) to CaGFP (15) and transformed the cassette into wild-type cells. Live-cell imaging of this transformed wild type showed a GFP signal representing active Rho1p in the cell periphery, which intensified at the emerging bud (Video S4a). Later, active Rho1p distribution constituted a gradient along the periphery of the daughter cell with increasing intensity toward the bud tip. This gradient faded progressively to disappear toward the end of the anaphase, where active Rho1p was evenly distributed along the cell periphery (Video S4a). During cytokinesis, it was highly localized to the site of cell division where a new septum was formed. Then, active Rho1p decreased to normal levels at the end of cell division. Likewise, we observed the same distribution during hyphal growth (Video S4b), as was also shown in previous studies (15).
Similarly, the Pkc1p-RBD-CaGFP cassette was transformed in wild-type or mutant backgrounds for coexpression either with Rho1p-13xcMyc, for pull-down assay, or with CaPHx2-CaRFP for subcellular colocalization using fluorescent imaging. Live-cell confocal imaging of either irs4 or inp51 mutants co-expressing CaPHx2-CaRFP and PKC-RBD-CaGFP showed that, in addition to the wild-type distribution of active Rho1p, there was an abnormal distribution that colocalized with aberrant PI(4,5)P2 patches (Fig. 4). The advent of this mislocalization happened mostly during the end of cytokinesis (Video S5). In addition to abnormalities in the distribution of active Rho1p, we quantitatively investigated active Rho1p in pull-down experiments followed by western blot in cells co-expressing RHO1-13xcMyc and PKC-RBD-CaGFP. As a negative control, we first used a strain that only expressed RHO1-13xcMyc. As expected, after pull-down with GFP-trap and western blot with α-Myc, no band could be detected (data not shown). Irs4 and inp51 co-expressing RHO1-13xcMyc and PKC-RBD-CaGFP showed an under-activation of Rho1p compared to wild type during the stationary phase (Fig. 5, top panel). On the other hand, the opposite was observed during the early exponential phase, as an overactivation of Rho1p was found in the mutants compared to wild type (Fig. 5, bottom panel). Overall, these findings imply that a disruption in localization and levels of PI(4,5)P2 in mutants is accompanied by a disturbance in Rho1p regulation and localization of its active form.
Fig 4.
Active Rho1p mislocalizes with the abnormal PI(4,5)P2 patches. Confocal microscopy inp51 (or irs4) mutant expressing CaPHx2-CaRFP and PKC-RBD-CaGFP. The left panel shows the RFP signal representing the distribution of PI(4,5)P2 in the PM, the center panel shows the GFP signal representing the distribution of active-Rho1p, and the right panel shows the merging of GFP and RFP signals, where the yellow signal represents the colocalization of PI(4,5)P2 and active Rho1p in the abnormal patches (depicted by white arrows; see Video S5).
Fig 5.
Mutants display an altered profile of Rho1p activation. Cells were grown overnight (stationary phase) or with an additional 4 hours of subculture (early exponential phase). After harvesting, cells were disrupted, and cell lysates with equal total protein content (200 µg) were incubated with anti-GFP (GFP-Trap, Chromotek) to pull down active Rho1p which binds PKC-RBD-CaGFP. A western blot using α-Myc was performed with pull-down eluate to detect active-Rho1p, or with 2 µg of total protein lysate to detect total Rho1p. The top panel shows a significant underactivation of Rho1p during the stationary phase, while the bottom panel shows a significant overactivation of Rho1p during the early exponential phase.
Similar defects related to PI(4,5)P2 are manifested during exposure of Candida wild-type cells to caspofungin
Our earlier study showed that wild-type C. albicans cells exposed to different concentrations of caspofungin rapidly mislocalized PI(4,5)P2 in a highly dynamic fashion and in a dose-dependent manner that correlated with fungicidal activity (7, 8). Caspofungin exposure not only disturbed PI(4,5)P2 distribution but also augmented PI(4,5)P2 levels over a 3-hour exposure experiment. Our earlier study showed some abnormal septation events and mislocalization of septins (Cdc10p and Sep7p) with PI(4,5)P2 (8). In this study, we exposed wild-type cells co-expressing CaPHx2-CaGFP and ACT1-CaRFP to 4× MIC caspofungin. Figure 6 shows that actin was recruited to a site of the PM where it seemed to be enriched with PI(4,5)P2. Furthermore, actin seemingly pulled the PM with PI(4,5)P2 inside the cell but later appeared to dissociate from the PM and PI(4,5)P2 (Fig. 6; Video S6). Likewise, wild-type cells co-expressing CaPHx2-CaGFP and MYO1-CaRFP mislocalized Myo1p along with PI(4,5)P2 (Fig. 7 and 8; Videos S7 and S8). Figure 7 shows that Myo1p mislocalized with PI(4,5)P2 starting at 9′ right at the site of cell division and after completion of cytokinesis (Video S7). The mislocalization started fading at 30′ when Myo1p dissociated from PI(4,5)P2. Figure 8 shows another type of defect caused by exposure to caspofungin, as Myo1p localization was normal during cell division from 5′ to 80′ (indicated with white arrow), while PI(4,5)P2 showed multiple dynamic mislocalizations. During the following cell division at 95′, the daughter cell from the previous cell division started a bud at a site (indicated by white arrow), but Myo1 is localized to a different site where another bud is started at 110′ (white arrowhead). The initial bud seemed to stall, but the second one continued the cell division with intermittent PI(4,5)P2 mislocalizations (Video S8). The figure and supplemental video also show a wider mother-daughter bud neck, a cellular abnormality we previously observed with exposure to caspofungin (8). Finally, Figure 9 shows dynamic mislocalization of both active-Rho1p and PI(4,5)P2, which together co-mislocalized to two different cellular localizations during a 6-minute interval. All these data show strikingly similar mislocalizations of key components of cytokinesis after deletion of either INP51 or IRS4 or after exposure to caspofungin. However, these phenotypes are not identical, as they are dynamic and transitory in the latter case.
Fig 6.
Exposure to the antifungal drug caspofungin mislocalizes actin along with PI(4,5)P2. Confocal live microscopy of wild-type cells co-expressing CaPHx2-CaGFP and ACT1-CaRFP, showing 3-minute time-lapse images after treatment with 4× MIC of caspofungin. The white arrow points to a PM location where Act1p is recruited at 3′ and continues to increase until 12′, after which the PM with its PI(4,5)P2 starts to be pulled inside the cell to create an invagination. At 21′ and 24′, Act1p seems to dissociate from the PI(4,5)P2 invagination (see Video S6).
Fig 7.
Exposure to the antifungal drug caspofungin mislocalizes Myo1 along with PI(4,5)P2. Confocal live microscopy of wild-type cells co-expressing CaPHx2-CaGFP and MYO1-CaRFP showing 3-minute time-lapse images after treatment with 4× MIC of caspofungin. The white arrowhead points to an instance of Myo1p mislocalization along with PI(4,5)P2, which happens right after the completion of cytokinesis (see Video S7).
Fig 8.
Exposure to the antifungal drug caspofungin mislocalizes Myo1p and causes other cellular defects. Confocal live microscopy of wild-type cells co-expressing CaPHx2-CaGFP and MYO1-CaRFP showing 5-minute time-lapse images after treatment with 4× MIC of caspofungin. The white arrow indicates Myo1p localization being normal during steps of this cell division from 5′ to 80′ (indicated with white arrow), while PI(4,5)P2 shows multiple dynamic mislocalizations. During the following cell division, at 95′, the daughter cell from the previous cell division starts a bud at a site indicated by the white arrow, while Myo1 is localized to a different site where another bud is started at 110′ (white arrowhead). The initial bud seems to stall, but the second one continues the cell division with intermittent PI(4,5)P2 mislocalizations (see Video S8). Note the unusually wide mother bud neck.
Fig 9.
Exposure to caspofungin mislocalizes active Rho1p along with PI(4,5)P2. Confocal live microscopy of wild-type cells co-expressing CaPHx2-CaRFP and PKC-RBD-CaGFP showing 3-minute time-lapse images at T 63′ and 66′ after treatment with 4× MIC of caspofungin. A dynamic mislocalization of active Rho1p together with PI(4,5)P2 is depicted in two different subcellular locations during a 6-minute period, indicated by the white arrowhead.
DISCUSSION
In previous work, we showed that exposure of C. albicans to caspofungin led to increased PI(4,5)P2 levels and septum-like invaginations of the PM, which contained PI(4,5)P2, septins, and cell wall and PM constituents. These subcellular phenotypes were accompanied by dose-response caspofungin fungicidal activity and paradoxical growth at concentrations up to 4× MIC and >4× MIC, respectively, and with levels of PKC-Mkc1 cell wall integrity pathway activation proportional to the degree of growth inhibition by the drug (8). The appearance of septation-like invaginations and the presence of broad-based C. albicans mother-daughter bud necks suggested that caspofungin exposure might be associated with disordered cytokinesis. In the present study, we link PI(4,5)P2 regulation more directly to cytokinesis and demonstrate that dysregulated PI(4,5)P2 and disordered cytokinesis are part of the natural response of C. albicans to caspofungin. Using live-cell imaging, we show that PM patches and invaginations that concentrate PI(4,5)P2 are correlated in time and space to sites of cytokinesis in C. albicans inp51 and irs4 mutant cells in which PI(4,5)P2 5-phosphatase activity is greatly attenuated. We then show colocalization of PI(4,5)P2 with Act1p, Myo1p, and activated Rho GTPase, the key components of the cytokinesis machinery. Finally, we demonstrate that cytokinesis is impaired in wild-type C. albicans SC5314 exposed to caspofungin, and PI(4,5)P2 patches colocalize with the same cytokinesis components. Our data, along with previous studies, support a model in which balanced PI(4,5)P2 regulation helps govern cytokinesis and cell wall integrity through a network that also includes septins and septin-regulating protein kinase Gin4, actin, myosin, Rho1, and the PKC-Mkc1 pathway (Fig. 10). Dysregulation of PI(4,5)P2 and this network, as seen with fungicidal caspofungin exposure and in inp51 or irs4 mutants, is associated with disrupted cytokinesis and reduced cell wall integrity.
Fig 10.
A schematic summary of the interplay between PI(4,5)P2 and Rho1p. At the PM, PI(4,5)P2 is synthesized from or degraded into phosphatidylinositol-4-phosphate (PI4P) by MSS4 or INP51, respectively. PI(4,5)P2 is able to interact with Rho1p either directly (Yoshida) or indirectly via ROM2 (Kobayashi). Interfering with this interplay between PI(4,5)P2 and Rho1p by either IRS4 or INP51 gene deletion, or by caspofungin exposure, leads to aberrant cytokinesis, perturbation in the cell-wall integrity pathway, and mislocalization of not only PI(4,5)P2 and Rho1p but also Act1p, septins, and Myo1p.
PI(4,5)P2, the most abundant among the seven phosphoinositides in eukaryotes, is localized to the PM, where it interacts with effector proteins to perform diverse cellular functions, including ion channel regulation, actin cytoskeleton remodeling, endocytosis, exocytosis, and phagocytosis. In addition, products of PI(4,5)P2 metabolism—conversion intophosphatidylinositol (3,4,5)-trisphosphate or degradation into inositol (1,4,5)-trisphosphate and diacylglycerol—also relay further functions (4, 11). Phosphoinositides could be thought of as phospholipidic anchors that are implicated in virtually any function that involves cellular membrane dynamics. The type of membrane to which each phosphoinositide localizes dictates the type of its functions. It is then no surprise that PI(4,5)P2 is an essential molecule for cell life, as is the gene encoding the protein that synthesizes PI(4,5)P2 from PI4P. This enzyme, which is a phosphatidylinositol 4-phosphate 5-kinase, was shown to be important for or regulate cytokinesis in Schizosaccharomyces pombe (its3 gene) (26, 27) and Drosophila melanogaster (fwd gene) (28). Several additional lines of evidence implicated PI(4,5)P2 in cytokinesis in various eukaryotes other than C. albicans. Overexpression of MSS4, an ortholog of its3, and Rho1 activation promotes cytokinesis, in the budding yeast Saccharomyces cerevisiae, even in the absence of an actomyosin contractile ring (29). Under normal circumstances, S. cerevisiae PI(4,5)P2 targets Rho1 to the site of cytokinesis, where the latter plays a central role in actomyosin ring assembly (29). Likewise, PLC activity is required for cytokinesis in flies spermatocytes from the order Diptera (30, 31). In mammalian cell lines, PI(4,5)P2 is enriched at the cleavage furrow and functions in the adhesion of the PM to the contractile ring (32). Sequestering PI(4,5)P2 or disturbing its level in eukaryotes perturbs cytokinesis (for a review see reference 12). This is the first study to explore associations between PI(4,5)P2, Rho1p, actomyosin ring, and cytokinesis in C. albicans.
We previously showed that PI(4,5)P2 patches colocalized with septins: Sep7p and Cdc10p (8). The role of septins (Sep7p, Cdc10p, etc.), actin (Act1p), and myosin-II (Myo1p) in cytokinesis is well established (33). It has been shown that PI(4,5)P2 can directly bind the N-terminus of septins to promote their filament organization and stability (34, 35), can affect actin polymerization, and modulate actin cross-linking and regulatory proteins (36). Indeed, the concentration of PI(4,5)P2 at the division site can facilitate the anchoring of structural components of the actomyosin ring to the PM (36). Work by Yoshida et al. suggests that PI(4,5)P2 could interact with a C-terminal polybasic sequence of Rho1p as part of a guanine nucleotide exchange factor-independent mechanism for targeting Rho1p to the bud neck (29). Our data present evidence that PI(4,5)P2 is not only a key player during cytokinesis, but its homeostasis is important for the correct spatial-temporal execution of this essential event in the cell cycle.
The echinocandin class of drugs, to which caspofungin belongs, impairs the synthesis of cell wall β−1,3-glucan (37) by inhibiting β−1,3-glucan synthase enzymes encoded by FKS genes (38). It is currently unclear how caspofungin elevates levels of PI(4,5)P2 and results in its mislocalization. Since the drug targets FKS genes, the most obvious links to PI(4,5)P2 are the Rho GTPase RHO1, which regulates FKS genes (39–42), or the RHO1 activator, the GDP/GTP exchange factor ROM2, which possesses a PH domain (data not shown) and has been shown in baker’s yeast to interact with PI(4,5)P2 (43). However, there are no data on possible mechanisms. It could also be a secondary effect to the cell wall stress imposed by caspofungin, or a direct effect of the drug on enzymes responsible for PI(4,5)P2 synthesis or degradation. Once PI(4,5)P2 is dysregulated, it is no surprise to see an effect on septins, Act1p, Myo1p, and active Rho1p, ultimately leading to defects in cytokinesis. We previously showed that caspofungin exposure rapidly causes a steady, dose-dependent increase in PI(4,5)P2 levels that continued throughout the 3-hour live-cell experiment and correlate with levels of fungicidal activity and paradoxical growth (8). PI(4,5)P2, septins, Act1p, Myo1p, and active-Rho1p mislocalization in wild-type cells exposed to caspofungin resembles that of inp51 and irs4 mutant cells in the absence of caspofungin exposure. However, these defects are transitory and more dynamic in caspofungin-treated wild-type cells. Taken together, our results allude to the possibility that the PI(4,5)P2 5-phosphatase encoded by INP51, along with its interacting partner Irs4p, is critical during mitotic exit when final cell division occurs, and that abnormal PI(4,5)P2 patches are remnants of imperfect cytokinesis. They also suggest that PI(4,5)P2, cytokinesis, and cell wall integrity pathway responses that are associated with deleterious outcomes of caspofungin exposure are overexuberant expressions of normally protective responses likely governed by the interplay between PI(4,5)P2 and Rho1p (Fig. 10).
Contributor Information
Hassan Badrane, Email: hab42@pitt.edu.
Andreas H. Groll, University Children's Hospital Münster, Münster, Germany
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/aac.01900-24.
Correlation in space and time between the appearance of the abnormal PI(4,5)P2 patches and cytokinesis, in irs4 and inp51 mutants. See Figure 1 for more details.
Mis-colocalization of Act1p (red signal) and the abnormal PI(4,5)P2 patches (green), in irs4 and inp51 mutants. See Figure 2 for more details.
Wild-type localization of Myo1p (green signal) and mislocalized Myo1p (red signal) along with abnormal PI(4,5)P2 patches (green), in the mutants. See Figure 3 for more details.
Normal localization of active form of Rho1p (green signal) in yeast and hyphal forms, respectively.
Mislocalization of active form of Rho1p (green signal) and the abnormal PI(4,5)P2 patches (red), in the mutants. See Figure 4 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mis-colocalization of Act1p (red signal) and the abnormal PI(4,5)P2 patches (green). See Figure 6 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mis-colocalization of Myo1p (red signal) and the abnormal PI(4,5)P2 patches (green). See Figure 7 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mislocalization of Myo1p (red signal), a defect in the choice of the bud site and an unusually wide budneck (green represents PI(4,5)P2). See Figure 8 for more details.
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Associated Data
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Supplementary Materials
Correlation in space and time between the appearance of the abnormal PI(4,5)P2 patches and cytokinesis, in irs4 and inp51 mutants. See Figure 1 for more details.
Mis-colocalization of Act1p (red signal) and the abnormal PI(4,5)P2 patches (green), in irs4 and inp51 mutants. See Figure 2 for more details.
Wild-type localization of Myo1p (green signal) and mislocalized Myo1p (red signal) along with abnormal PI(4,5)P2 patches (green), in the mutants. See Figure 3 for more details.
Normal localization of active form of Rho1p (green signal) in yeast and hyphal forms, respectively.
Mislocalization of active form of Rho1p (green signal) and the abnormal PI(4,5)P2 patches (red), in the mutants. See Figure 4 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mis-colocalization of Act1p (red signal) and the abnormal PI(4,5)P2 patches (green). See Figure 6 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mis-colocalization of Myo1p (red signal) and the abnormal PI(4,5)P2 patches (green). See Figure 7 for more details.
Wild-type SC5314 <i>C. albicans</i> cells exposed to 4xMIC of caspofungin show mislocalization of Myo1p (red signal), a defect in the choice of the bud site and an unusually wide budneck (green represents PI(4,5)P2). See Figure 8 for more details.