Fluorescent Tracking of the Endoplasmic Reticulum in Live

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Fluorescent Tracking of the Endoplasmic Reticulum in Live Pathogenic Fungal Cells Raphael I. Benhamou,a,|| Qais Z. Jaber,a,|| Ido M. Herzog,a Yael Roichman,a and Micha Fridmana,*

a

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv

University, Tel Aviv, 6997801, Israel.

KEYWORDS: Fluorogenic probes, Organelle trackers, Live cell imaging, Antifungal azoles.

ABSTRACT In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this

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organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered as the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live

Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed contact sites between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.

INTRODUCTION

Epifluorescence and confocal microscopy in combination with organelle-specific fluorescent dyes are amongst the most common and broadly applied tools for studying biological processes in living cells.1–3 The cytoplasm of eukaryotic cells is embedded with organelles that have different essential roles in cellular functions. Organelle-specific


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dyes, also termed organelle trackers, can be used as counterstains to help identify the location of a target protein that is fluorescently labeled by a reporter fluorescent protein or by a specific fluorescent antibody.4,5 These imaging tools are also essential for studying the functions and dynamics of the different organelles during the cell cycle, for studying inter-organelle interactions and organization, and for evaluating the effects of small molecules such as drugs, nutrients, and metabolites on the assembly and functions of specific organelles. Numerous fluorescent organelle trackers have been developed for use in biological and medical research as well as for diagnostic applications during recent decades.6–8 The choice of a fluorescent tracker is based on spectral properties such as excitation and emission wavelengths, stability to bleaching, cell toxicity, and specificity for the organelle of interest.9,10 Notably, the outstanding diversity of eukaryotic cells in nature is manifested in structural and functional differences that can greatly affect the performance of an organelle tracker. For example, the suitability of an organelle-specific tracker that is based on binding to a specific protein that localizes to the target organelle depends on the level of its expression and subcellular distribution in the specific cell investigated. Other variable cellular


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characteristics that can perturb the organelle-specificity of an organelle tracker involve the expression of efflux pumps that reduce the concentration of the dye in the cell, cell permeability, and off-target binding sites.2,11,12 Most commercially available organelle trackers were developed and optimized for use in mammalian or plant cells; there are relatively few examples of trackers that specifically label organelles in fungal cells.

In recent decades, health organizations around the globe have reported an alarming increase in the percentage of serious infections that are caused by drugresistant pathogenic fungi.13,14 Members of the Candida genus, including C. albicans and C. glabrata and, in recent years, the highly drug resistant C. auris, are the most commonly encountered fungal pathogens that cause high mortality rates, especially amongst patients with a compromised immune system.15–17 Unfortunately, the urgent need to develop novel and more effective antifungal drugs is confounded by the very limited number of drug targets available in the fungal cell as a result of the evolutionary similarity between fungal and mammalian cells. The predominant target for antifungal drug development is ergosterol, an essential sterol component of the fungal cell plasma


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membrane, and its biosynthetic pathway. Ergosterol has the same functions in the fungal membrane as cholesterol has in the plasma membrane of mammalian cells. Antifungal azoles that are used as first-line drug treatment for fungal infections inhibit activity of CYP51, a cytochrome P450 lanosterol 14α-demethylase that is involved in the biosynthesis of ergosterol.18,19 Like several other enzymes involved in ergosterol biosynthesis, CYP51 localizes primarily to the endoplasmic reticulum (ER).20 As the ER is the main location of ergosterol biosynthesis, visual information about the assembly, shape-dynamics and components of the ER in cells of fungal pathogens would be of great value; however, no examples of ER-specific trackers optimal for use in pathogenic yeasts have been made available to date.

The dicarbocyanine dye DiOC6 was previously reported to stain the ER of cells of the baker’s yeast Saccharomyces cerevisiae; however, DiOC6 non-specifically labels intracellular membranes, and it is also cytotoxic.21,22 Moreover, depending on the concentration and on the specific strain, DiOC6 was shown to label either the ER or the mitochondria of the yeast cell. Two other ER trackers that were developed for


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mammalian cells, a BODIPY-based brefeldin A derivative and the dapoxyl-based BlueWhite DPX, were used for ER labeling in hyphal tips of the tree fungus Pisolithus

tinctorius.23,24 Blue-White DPX was also used for ER labeling of Aspergillus oryzae, a filamentous fungus used for fermentation of soybeans, and of S. cerevisea.25,26

In this study we report on the development of trackers that effectively and specifically stain the ER of a collection of pathogenic Candida yeast cells. In designing fluorescent fungal-specific ER trackers we focused on the pharmacophore of the antifungal drugs fluconazole and itraconazole (Figure 1). Recently, using fluorescent antifungal azoles, we showed that the subcellular distribution of these drugs can be altered by attaching different fluorescent dyes to the pharmacophore structure of fluconazole. Attachment of dansyl or Cy5 dyes results in antifungal azoles that localize mainly to mitochondria of Candida yeast cells during the first few hours of exposure.27–29 On the other hand, attachment of a 7-(diethyl)-aminocoumarin dye to the pharmacophore of fluconazole (compound 1, Figure 1) directed the drug mainly to the ER.30 Compared to the mitochondria-localized azoles, ER-localized azole 1 displayed


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up to two orders of magnitude improvement in antifungal activity and reduced the growth of drug-tolerant fungal subpopulations in a panel of Candida.30 We also reported the synthesis of compound 2 (Figure 1), the 1,2,3-triazole derivative of antifungal azole 1 that localized to the ER of Candida cells and displayed no antifungal activity.30

O N

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Figure 1. Antifungal azole drugs fluconazole, itraconazole, and fluorescent fungal-ERlocalizing azoles 1-6. The 1,2,4-triazole rings are colored red, 1,2,3-triazole rings are colored blue, fluorides are colored green, and chlorides in orange.


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RESULTS AND DISCUSSION

Using the pharmacophores of fluconazole and itraconazole (Figure 1), we designed and synthesized fluorescent azoles 3-6 and evaluated their localization in a collection of pathogenic yeast cells (for synthetic procedures see supporting information sections 1.4 and Supplementary Schemes S1, S2, and S3). To be effective for use in living cells, a tracker must not affect cell viability at labeling concentration and during the time of the experiment. To reduce fungal cell toxicity, azoles 2, 3, 5, and 6 were designed with a 1,2,3-triazole instead of a 1,2,4-triazole ring. In triazole-based antifungal azoles, the 1,2,4-triazole ring interacts with the iron atom in the heme of the target CYP51. Through density functional theory calculations, we previously showed that the electron density of the nitrogen atom in 1,2,4-triazoles that interacts with the heme iron is 33% higher than that of the corresponding nitrogen in 1,2,3-triazoles.30 We therefore reasoned that isosteric 1,2,3-triazole-based analogs of antifungal azoles bind in the catalytic domain of CYP51 to facilitate specific ER-labeling in fungal cells but would not have a significant effect on the catalytic activity of the enzyme. The


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difluorophenyl ring of segment of azoles 1, 2, 4, and 5 was displaced by dichlorophenyl in azoles 3 and 6 to study possible halogen atom effects on fluorescence, localization, and cell permeability. To extend compatibility of the ER trackers with other fluorescent markers by preventing excitation/emission overlaps, compounds 1-3 are based on 7(diethyl)-aminocoumarin, whereas compounds 4-6 are BODIPY-based.

We compared absorption and emission spectra of fluorescent azoles 1-6 in aqueous PBS buffer solution at pH 7.4 (Supporting Information section 1.5). Interestingly, although aminocoumarin-based azoles 1-3 shared the same absorption spectrum maxima value (430 nm), their emission spectra and maxima values differed significantly (Supplementary Figures S1-S4). In ethanol, the emission maxima values (470 nm) were similar, suggesting that the measured differences in aqueous buffer solution likely resulted from aggragation (Supplementary Figures S5-S8). In contrast no differences in emission maxima values were observed for the BODIPY-based azoles 46; these three compounds shared the same absorption (505 nm) and emission (515 nm) maxima values (Supplementary Figures S9-S12).


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ER-labeling properties were evaluated by incubating the compounds with representative strains of C. albicans and C. glabrata, considered the two most common fungal pathogens that cause infections in humans. These two types of Candida are phylogenetically, genetically, and phenotypically different.31 Since our goal was to develop ER trackers suitable for staining of live cells, we first tested if the synthetic fluorescent azoles displayed antifungal activity against the panel of Candida by determining their minimal inhibitory concentration (MIC) values (Supporting Information section 2.1 and Supplementary Table S1).32 The aminocoumarin-based azole 1, which has a 1,2,4-triazole ring, had potent antifungal activity against all tested Candida strains with MICs ranging from 0.007 to 16 μg/mL. The BODIPY-based azole 4, which has a 1,2,4-triazole ring, had MICs of 0.5-1 μg/mL against the tested C. albicans strains but was inactive against the tested C. glabrata strains (MIC≥ 64 μg/mL). Azoles 2, 3, 5, and 6, that contain a 1,2,3-triazole ring, had no antifungal activity at the highest concentration tested (MIC ≥ 64 μg/mL). These azoles were therefore further evaluated as fungal ER trackers in live cell imaging experiments with four Candida strains (Figure 2 and Supplementary Figures S13-S16). As control dyes we tested the commercial ER-


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trackers BODIPY TR Glibenclamide33 (ex: 587 nm; em: 615 nm), Blue-White DPX34 (ex: 374 nm; em: 430-640 nm) and DiOC622 (ex: 480 nm; em: 500 nm).

In yeast cells, the ER forms a distinct circular pattern around the nuclear envelope that extends in branches that occupy the cytoplasm; these branches are termed the peripheral ER. As in cells of higher eukaryotes, in yeast cells, the peripheral ER is mainly organized into a membrane network adjacent to the inner leaflet of the plasma membrane; the ER region near the plasma membrane is referred to as the cortical ER.35 To optimize the probes we investigated the effects of the concentration, time of incubation, and staining medium (for optimal staining conditions, see the Experimental Section). In C. albicans cells incubated with the commercial ER tracker Blue-White DPX, we observed non-specific staining of the cytoplasm (Figure 2A-B and Supplementary Figure S15). Blue-White DPX did, however, label the ER in yeast cells of the C. glabrata strains in our panel (Supplementary Figures S13, S14). Although a staining pattern expected for the ER was observed when DiOC6 was incubated with C.

albicans strain SN152, non-specific staining of other cellular compartments such as lipid


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droplets was also observed (Figure 2C-D). Non-specific labeling of organelles by DiOC6 was more significant in C. albicans than in C. glabrata yeast cells (Figure 2D and Supplementary Figures S13-S15).

Figure 2. Localization of azoles 2, 3, 5, and 6 and commercial ER trackers in C.

albicans SN152. Cells were incubated for 60 min with A-B) Blue-White DPX (red, 1 μM), C-D) DiOC6 (yellow, 1 μg/mL), E-F) azole 2 (cyan, 10 μM), G-H) azole 3 (cyan, 10 μM), I-J) azole 5 (green, 10 μM), K-L) azole 6 (green, 10 μM). For each dye, the brightfield image is shown on the left and the fluorescent image on the right. The bandpass filters


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used to image Blue-White DPX were ex: 390 nm and em: 525 nm; to image DiOC6, azole 2, and azole 3 were ex: 440 nm and em: 480 nm; and to image azole 5 and azole 6 were ex: 470 nm and em: 525 nm. Scale bars, 5 µm. Under the conditions screened, the commercial ER tracker BODIPY TR Glibenclamide did not stain the cell interior of any of the tested Candida strains (Supplementary Figure S19). When cells of the tested panel of Candida strains were stained with azoles 2, 3, 5, and 6 the distinct nuclear envelope and cortical structure of the ER were clearly observed (Figure 2E-L and Supplementary Figures S13, S14 and S15). Finally, the ethyl ester of 7-(diethyl)-aminocoumarin, which is the fluorophore segment of azoles 1-3, did not stain the yeast cell interior of any of the tested Candida strains (Supplementary Figure S20); in yeast cells labeled with only the BODIPY-methyl ester, the fluorophore segment of azoles 4-6, the entire cytoplasm was labeled in a non-specific pattern (Supplementary Figure S21). This demonstrates that ER-labeling specificity of the fluorescent azoles does not result from their fluorescent dye segment.

Since an ER-pattern was observed in C. glabrata yeast cells that were stained with Blue-White DPX but not in C. albicans, we treated C. glabrata with azole 5 and ER-


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tracker Blue-White DPX; azole 5 was chosen since the excitation/emission wavelengths of this compound and of Blue-White DPX do not coincide. In C. glabrata cells stained with Blue-White DPX, a circular pattern around the nuclear envelope with peripheral cortical extensions reminiscent of the ER structure was clearly visible (Figure 3A-B). High co-localization was observed between the subcellular staining patterns of azole 5 and that of the Blue-White DPX (Figure 3A-D) with a Pearson correlation coefficient36 of 0.86±0.04 further supporting the ER-selectivity of the fluorescent azoles. Next, we determined the localization of azole 5 in yeast cells of a C. albicans strain that expresses mCherry-labeled Eno1, a protein that localizes largely to the nucleus.37 The fluorescent pattern of azole 5 surrounded the Eno1-mCherry-labeled nuclei as expected of an ER localization which circles the nuclear envelope (Figure 3E-H and Supplementary Figures S17, S18).


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Figure 3. A-D) Images of yeast cells of C. glabrata strain 2001 that were incubated with Blue-White DPX (red, 1 μM) and azole 5 (green, 10 μM) for 60 min. E-H) Images of yeast cells of C. albicans expressing Eno1-mCherry that localizes mainly to the nucleus (red) were incubated with azole 5 (green, 10 μM) for 60 min. I-J) Images of yeast cells of C. albicans SN152 were incubated with azole 5 (green, 10 μM) for 60 min. collected K-L) Images of yeast cells of C. albicans erg11ΔΔ/erg3ΔΔ were incubated with azole 5 (green, 10 μM) for 60 min. Scale bars, 5 µm.


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To further study the ER specificity of the fluorescent azoles, we compared the labeling patterns in a C. albicans mutant strain lacking copies of both the ERG11 and

ERG3 genes to that of strain SN152 from which the mutant strain was derived (Supplementary Table S2). ERG11 encodes CYP51, the target of antifungal azoles that is essential for fungal cell growth.38 ERG11, which encodes CYP51, the target of antifungal azoles, is essential for fungal cell growth under aerobic conditions when

ERG3, which encodes a C-5 sterol desaturase, is functional. Therefore, an erg11∆∆ erg3∆∆ mutant strain, which is viable despite the absence of CYP51, was used to study the ER selectivity of the fluorescent azoles. While the characteristic nuclear envelope and cortical ER pattern appeared in cells of the parental C. albicans SN152 strain that labeled with azole 5, no ER-labeling pattern appeared in cells lacking CYP51 and the entire cytoplasm of these cells was stained (Figure 3K-L and Supplementary Figure S22). This supports that the reason for the ER specificity of the azole fluorescent probes results from the pharmacophore of the azole class of antifungal drugs that binds to CYP51, which localizes primarily to the ER in yeast cells.


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With the reported fluorescent ER trackers it should be possible to detect interconnections between the ER and other organelles by simply staining the fungal cells with a combination of organelle trackers. This strategy offers a robust protocol for studying organelle interconnections and saves the need for cloning cells with fluorescent reporter proteins. It is now well accepted inter-organelle communication occurs via membrane contact sites (MCSs).39 At these sites organelles are tethered together in close proximity to facilitate various inter-organelle functions. MCSs between the ER and mitochondria are involved in calcium signaling, lipid biosynthesis, and mitochondrial division.40,41 Contacts between the ER and mitochondria were previously visualized by confocal fluorescence microscopy in live cells.42 In these studies, fluorescent labeling of the ER and mitochondria was accomplished using organellespecific fluorescent reporter proteins and/or immunoblotting.42,43 We demonstrated that visualization of the intracellular organization of the ER and mitochondria and the identification of MCSs between these two organelles in Candida cells were possible by staining of the cells with a combination of ER-dye 3 and a mitochondrial dye composed of a Cy5-based antifungal azole previously developed in our group (Figure 4A-B).27


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Figure 4. MCSs between the ER and mitochondria visualized by fluorescence microscopy in live C. albicans SN152 cells. A) Representative images of Cy5-based mitochondrial dye (red) after excitation at 560/25 nm; emission was monitored at 684/24 nm. B) Representative images of azole 3 (cyan) after excitation at 427/10 nm; emission was monitored at 510/20 nm. C) Merged images. Arrowheads mark sites of colocalization. Lower panels are zoom-in of regions boxed in upper panels. Cells were incubated for 5 min with 1 µg/mL Cy5-based mitochondrial dye and 10 µM of 3. Images were

processed

by

convolution with

a

Gaussian function for

smoothing

using

ImageJ program.

Merged images of the fluorescently labeled mitochondria and ER revealed sites that can indicate on the localization MCSs where the characteristic uniform network of


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mitochondrial tubules co-localizes with the ER (Figure 4C). Similar co-localization sites were previously observed between GFP-labeled ER and RFP-labeled mitochondria in yeast cells of Saccharomyces cerevisiae using confocal microscopy and were attributed to tethering sites between the two organelles.44

CONCLUSION

To conclude, the reported azole-based fluorescent probes proved effective and specific ER trackers in our analysis of a panel of fungal pathogens from the Candida genus, the most common causes of fungal infections in humans worldwide. These ER trackers displayed several superior properties compared to commercially available ER trackers: Compared to the commercial trackers, the azole-based probes specifically stained the ER with less background signal. The commercial ER-tracker BODIPY TR Glibenclamide did not stain any of the strains in the tested Candida panel, and the commercial ER-tracker Blue-White DPX stained only C. glabrata cells. In contrast, all the reported fluorescent azole probes effectively stained the ER of all of the strains in


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the panel. No non-specific labeling of additional intracellular compartments, such as lipid droplets, was observed in cells that were stained with any of the reported fluorescent azoles; such non-specific labeling was observed in cells stained with BlueWhite DPX and the dicarbocyanine-derived ER-tracker DiOC6. Whereas DiOC6 is known to cause cytotoxic effects at concentrations used for ER labeling, fluorescent probes 2, 3, 5, and 6, which contain 1,2,3-triazole rings, did not display antifungal activity at concentrations significantly higher than those used for ER labeling and are therefore suitable for live cell imaging experiments. Finally, we demonstrated simultaneous labeling of both the ER and the mitochondria of C. albicans cells using a combination of an ER tracker described here and a mitochondrial tracker. Using these two trackers, it was possible to identify inter-organelle organization and co-localization sites that can be attributed to membrane contact sites between the two organelles in live Candida cells. Thus, the reported trackers offer robust new molecular tools for the study of the ER and its interconnections with other organelles in live cells of important fungal pathogens.


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EXPERIMENTAL SECTION Preparation of stock solutions of the tested compounds. All fluorescent probes were dissolved in DMSO to final concentration of 5 mg/mL. The antifungal drugs fluconazole (FLC) and itraconazole (ITR) were purchased from Sigma Aldrich. FLC was dissolved in anhydrous ethanol and ITR in DMSO to a final concentration of 5 mg/mL. ER-tracker DPX and DiOC6 were purchased from Thermo Fisher and BODIPY™ TR Glibenclamide was purchased from Setareh BioTech. BODIPY™ TR Glibenclamide and ER-tracker DPX were dissolved in DMSO to a final concentration of 1 mM. DiOC6 was dissolved in DMSO to a final concentration of 1 mg/mL.

Minimal inhibitory concentration broth double-dilution assay. All strains (Supplementary Table S1) were tested using the double-dilution method in 96-well plates (Corning). All of the yeast strains in this study were grown in casitone growth medium. Starter cultures were incubated for 24 h (37 °C, 5% CO2, aerobic conditions) and then diluted 1:100 into fresh medium. Compounds dissolved in ethanol or in DMSO were added to the casitone broth to form the mother liquor (32 μL stock solution in 1218 μL of casitone) at the


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starting concentration of 64 μg/mL. Next, 100 μL of serial double dilutions of compounds in Casitone (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.014, and 0.007 μg/mL) were prepared in flat-bottomed 96-well microplates (Corning). Control wells with no compounds and wells without yeast cells containing each tested concentration of the compounds (blanks) were also prepared. An equal volume (100 μL) of yeast suspensions in Casitone broth that was prepared as follows: 900 mL doubly distilled H2O, 9 g Casitone (bacto-casitone), 5 g yeast extract, 11.5 g sodium citrate dihydrate, 20 g glucose. The yeast suspension was added to each well for a final volume of 200 μL. The ethanol or the DMSO concentrations ranged from 0.0012% to 1.3%. The final inoculum was between 5 x 104 CFU/mL and 5 x 105 CFU/mL; identical results were obtained when using 5 x 103 CFU/mL as a final inoculum. After incubation for 24 h at 37 °C in 5% CO2, MTT (50 μL of a 1 mg/mL solution in H2O) was added to each well followed by additional incubation at 37 °C for 2 h. Results were confirmed in two independent experiments, and each concentration was tested in triplicate.


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Growth conditions for live cell imaging. Cells were grown to log phase in liquid YPD overnight at 30 °C in a 10 ml tube. Cells were diluted 1:10 and then incubated for 2 h at 30 °C to log phase.

Cell staining. The fluorescent probes were added to a final concentration of 10 μM for azoles 1-6 to cells from a log phase culture grown in YPD broth media at 30 °C. Cells were then incubated in the dark for a total of 60 min. Before imaging, cells were washed once with PBS buffer. For ER-tracker dyes, DiOC6 (1 μg/mL) and BODIPY™ TR Glibenclamide (1 μM), staining cells were incubated in a prepared Hanks' Balanced Salt Solution at 37 °C for 5 min and 30 min, respectively. For ER-tracker Blue-White DPX (1 μM), staining cells were incubated in YPD for 30 min at 30 °C. For mitochondrial staining the previously reported Cy5-based mitochondrial dye was used (1 μg/mL). For staining, cells were incubated in YPD in the presence of dye for 5 min at 30 °C. Cells were then washed with PBS buffer once.

Live cell imaging. Candida cells were resuspended in PBS buffer and 2 μL were placed on a glass slides and covered with glass coverslips. C. glabrata (ATCC 2001) cells


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treated with azoles 1-6, all tested Candida cells treated with ER-tracker Blue-White DPX and inter-organelle interaction study were imaged on a MORE imaging system (TILL Photonics GmbH) with an Olympus UPlanApo 100X 1.3 NA oil immersion objective. The bandpass filter sets used to image azoles 1-3 were excitation 427/10 nm and emission 510/20 nm. The bandpass filter sets used to image azoles 4-6 were excitation 485/20 nm and emission 525/30 nm. The bandpass filter sets used to image ER-tracker BlueWhite DPX were excitation 390/40 nm and emission 525/30 nm. The bandpass filter sets used to image Cy5-based mitochondrial dye were excitation 560/25 nm and emission 684/24 nm. C. albicans cells (ATCC 24433 and SN152) and C. glabrata (ATCC 66032) treated with azoles 1-6 were imaged on a Nikon Ti microscope equipped with a Plan Apo VC 100X oil objective and a Zyla 5.5 sCMOS camera (Andor) run by NIS elements Ar software. The bandpass filter sets used to image azoles 1-3, and DiOC6-stained cells were excitation 440 nm and emission 480/40 nm. The bandpass filter sets used to image cells treated with azoles 4-6, excitation 470 nm and emission 525/50 nm were used. For Eno1-mCherry, excitation 585 nm and emission 630/75 nm were used. To optimize the probes we investigated the effects of the concentration (1, 5


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and 10 μM), time of incubation (5, 30, 60 and 120 min) and two different media (YPD and HSSB). Images were processed using ImageJ program. The smoothed imaged were generated using the convolve filter (Normalize Kernel).

ASSOCIATED CONTENT

Supporting Information. Detailed synthetic procedures;

1H,

19F,

and

13C-NMR

assignments and spectra; High resolution MS data; Absorption/Emission spectra; yeast strains; MIC table and live cell imaging data. This material is available free of charge via the Internet.

AUTHOR INFORMATION

Corresponding Author * [email protected]

Author Contributions

|| These

authors contributed equally


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources This work was supported by Israel Science Foundation Grant 6/14 (Micha Fridman).

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

We thank Y. Ebenstein and J. Berman for granting us access to their fluorescent microscope systems.

ABBREVIATIONS CYP51: cytochrome P450 lanosterol 14α-demethylase; Cy5; cyanine 5; DiOC6: dicarbocyanine dye; ER: endoplasmic reticulum; FLC: fluconazole; ITR: itraconazole; MIC: minimal inhibitory concentration; MCS: membrane contact site.

REFERENCES


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(1)

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TABLE OF CONTENT ART WORK


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Fluorescent Tracking of the Endoplasmic Reticulum in Live

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