Measuring Replicative Lifespan in Cryptococcus neoformans

Abstract

Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.

1 Introduction

Replicative lifespan (RLS) depends on the number of cell cycle progressions and is defined as the number of daughter cells a mother cell produces before reaching senescence and death [1]. In the budding pathogenic yeast Cryptococcus neoformans, the asymmetric division ensures that the mother cell retains the classic aging-associated phenotypes, such as a decrease in vacuole acidity [2], increase in cell size [3], and cell wall thickness [2]. Investigating the impact of genetic, pharmacological, and metabolic modifications on controlling RLS [1, 4] constitutes a valuable tool for studying the aging process in yeast.

Currently, two methods are available to determine the RLS of C. neoformans cells: microdissection and using a microfluidic system [5, 6]. The first approach consists of manually separating the cells after each division and is the most commonly applied assay to determine the life span of a particular strain (Fig. 1) [7]. However, this traditional assay is labor-intensive and time-consuming. Other disadvantages include the reduced sample size that may not accurately estimate the stochasticity of the RLS and the limited number of strains that can be analyzed simultaneously [8, 9]. Also, this system does not allow the characterization of the cells at the molecular level. Therefore, an automated microfluidic tracking system was developed that enables more cells to be evaluated at once in a reduced time frame. By using a microfluidic system combined with time-lapse microscopy for RLS characterization, it is possible to verify morphological and phenotypical changes, including protein expression and localization, at a single cell level during aging (Fig. 2) [4, 6].

Fig. 1

Traditional microdissection workflow. (a) Inoculate Cryptococcus neoformans cells as a single thick line at the top of the desired agar plate using a sterile inoculation loop. (b) Once cells have grown, place the plate in the microscope and drag cells to the side using the fiber optic needle until single cells can be isolated. (c) Array 20–30 single cells in a line. (d) Once the mother cells generate a naïve cell, separate the cells, keeping the naïve cells in a line and dragging the mother cells away. The naïve cells will now be monitored as the mother cells. (e) Each time the mother cells bud, separate and count the daughter cells, dragging them away from the line. (f) Incubate the plate at 37 °C between budding events and store it at 4 °C overnight. (g) Terminate the study when cells have reached senescence and have not divided over 24 h. (h) Plot and analyze the data. The illustration was created using BioRender

Fig. 2

Scheme of the microfluidic system. (a) High-throughput Yeast Aging Analysis for Cryptococcus (HYAAC) device. Channel has two upper inlets, one long channel, and a single outlet. (b) Microscopic image of yeast cells growing with age in buckets arrayed in channel while daughter cells are washed away. Blue arrow represents media flow direction. (c) Pump with larger syringe containing media flowing through tubing into one of the inlets of the device. (d) HYAAC device is set on inverted microscope stage. Inoculum is loaded using small syringe into the other upper inlet. Tubing is also connected to the outlet, which allows media to flow from the outlet into a waste container. (e) Images of Cryptococcus RLS are captured over time and displayed on the computer monitor

The analysis of longevity in individual single yeast cells advances our understanding of the stochasticity of regulatory pathways that evolutionarily contribute to the mechanisms of aging [4]. Studying the RLS of pathogenic yeasts may shed light on important metabolic pathways and protein expression that directly promotes selective pressure leading to the accumulation of old cells, increasing tolerance to treatment, and the persistence of fungal infections [5, 6, 10]. In this chapter, we describe in detail these two procedures for quantifying the RLS of C. neoformans cells.

2 Materials

2.1 Traditional Microdissection

• 1. Yeast peptone dextrose (YPD) agar media: 1% yeast extract, 2% peptone, 2% glucose, 2% agar.

• 2. Synthetic media: 1.7 g yeast nitrogen base without amino acids, 1 g drop out mix, 4 mL ethanol, 5 g (NH₄)₂SO₄, 3.3 g NaCl, 2% glucose.

• 3. The desired media for microdissection supplemented with 2% agar (see Note 1).

• 4. Petri dishes (100 mm × 15 mm), sterile.

• 5. Loop 1 μL, sterile.

• 6. C. neoformans frozen stock.

• 7. Pipettes 25 mL and 50 mL.

• 8. Incubator at 37 °C.

• 9. Refrigerator at 4 °C.

• 10. Fiber optic needle (25 μm) on a tetrad dissection Axioscope A1 microscope.

• 11. GraphPad Prism software.

2.2 Microfluidic System

• 1. C. neoformans frozen stock.

• 2. Culture media (e.g., synthetic medium, SD).

• 3. Glass Erlenmeyer flask 250 mL, sterile.

• 4. Shaking incubator at 37 °C with 150 rpm.

• 5. Pipettes 50 mL, sterile.

• 6. Phosphate buffered saline (PBS).

• 7. Centrifuge.

• 8. Hemocytometer.

• 9. High-Throughput Yeast Aging Analysis for Cryptococcus (HYAAC) device: A polydimethylsiloxane (PDMS) chip bonded to a glass slide having a single channel (height 10–12 μm) with two inlets, one middle long channel (1 cm), and one single outlet (Fig. 2a). Each channel is comprised of 80 rows of staggered buckets that alternate between six or seven buckets per row (see Note 2). The buckets are modified triangles designed to be facing each other (Fig. 2B), where the bottom of the bucket is 3 μm wide and the top of the bucket is 9 μm wide (see Note 3).

• 10. Syringes (1 mL and 30 mL) with Luer-Loks to prevent leakage (see Note 4).

• 11. Tygon microbore tubing 0.020“ × 0.060” outer diameter (OD).

• 12. Tubing adapter 23G.

• 13. Metal pin 0.025″ × 0.05″.

• 14. Metal tubes 0.025 OD × 0.017 inner diameter (ID) × 0.5″ (see Note 5).

• 15. Syringe pump, with multi-syringe capacity and speed.

• 16. Waste collector (Flask).

• 17. Inverted fluorescent microscope with slide adapter, 40× objective lens, automated stage with x, y, and z directions, temperature-controlled and attached camera to record images at regular intervals over time (see Note 6).

• 18. Fiji software to process the images.

3 Methods

3.1 Traditional Microdissection

• 1. To make the stock agar plates, aliquot approximately 15 mL of YPD agar media in each plate. After media solidification, recover and streak the desired yeast strain from a – 80 °C cryovial using a sterile loop onto the YPD agar plate.

• 2. Incubate for 48 h at 37 °C.

• 3. For making the microdissection plates, aliquot 35 mL of desired media with 2% of agar into each Petri dish (see Note 7).

• 4. Using a sterile loop, pick the desired yeast strain from the stock plate and streak it on the microdissection plate agar as a single thick line on the top part of the agar (Fig. 1a).

• 5. Incubate plates for 48 h at 37 °C, allowing growth of the cells.

• 6. On a tetrad dissection Axioscope A1 microscope (Zeiss) at 250× magnification, adjust the position of a 25 μm fiber optic needle so that it is located in the center of the microscope field of view.

• 7. Move the needle upward by lowering the joystick. The tip of the needle should touch the agar without perforating it.

• 8. To transfer the C. neoformans cells, very gently move the needle upward into the cell growth and drag it to the side while still in contact with the agar. Repeat the action until single cells can be separated (Fig. 1b).

• 9. Array 20–30 cells in a line in the center of the plate (Fig. 1c).

• 10. Allow at least 50 μm between each cell to ensure enough space to manipulate neighboring cells (see Note 8).

• 11. The first bud of each cell is identified as a naive cell. Keep the naive cell and drag the mother cell away from your line of arrayed cells (see Note 9). The bud cells can be separated by touching the needle to the bud and dragging it away from the mother cell. Alternatively, the needle can be positioned to touch both the mother and the bud cell and moved around slightly with gentle taping to the joystick to promote separation (Fig. 1d).

• 12. This naive daughter cell is now the mother cell that will be observed throughout the experiment.

• 13. New buds from the mother cell are separated at the end of each division (1–2 h). From this moment, each daughter cell separated from the mother should be counted and recorded as one generation (Fig. 1e) (see Note 10).

• 14. Between separations, the plate is returned to the incubator at 37 °C or maintained at 4 °C overnight to prevent excessive budding (Fig. 1f) (see Note 11). Seal the plates with parafilm to avoid contamination.

• 15. A cell is considered dead when it has not divided for a period of at least 24 h, with incubations at 37 °C (see Note 12).

• 16. Terminate the study when over 50% of arrayed cells have reached senescence. The RLS of each cell is the sum of the total budding events until the mother cell’s death (Fig. 1g).

• 17. Plot and analyze the data using GraphPad Prism software (Fig. 1h). Data may be plotted as a survival graph (% survival vs. generations), where the Wilcoxon rank sum test is used to calculate p values. Alternatively, data can be plotted as a box or violin column graph (generations vs. strains), where a Student’s t-test is used to calculate p values.

3.2 Microfluidic System: HYAAC

• 1. Start an overnight Cryptococcus culture at 37 °C and 150 rpm in desired liquid media (see Note 13). The next day, subculture cells 1:50 in a new flask using the same conditions. Grow Cryptococcus for 6 h to ensure a young, exponential culture (approximately 2 generations).

• 2. Centrifuge cells at 4000 × g for 5 min and wash yeast cells with PBS twice. Dilute Cryptococcus cells to a concentration of 10⁵ cells/mL and load the suspension into a 1 mL syringe. Load a larger syringe with media.

• 3. Attach both syringes to a tubing adapter and connect each of them to Tygon microbore tubing. Then, insert a metal tube to the end of each Tygon microbore tubing.

• 4. Connect a metal tube to the outlet of the HYAAC system, attaching a Tygon microbore tubing to the end of the metal tube.

• 5. Load the media-filled syringe to the syringe pump and attach the end of the metal tube to one of the inlets of the HYAAC device. Pump media into the system at a constant rate of 10–15 μL/min until it fills the entire device and starts to come out of the outlet tubing into a waste container (Fig. 2c, d).

• 6. Pause the syringe pump to hold the pressure within the system and place the device atop an onstage incubator of an inverted microscope that keeps the chip and its contents at 37 °C (Fig. 2d).

• 7. At the other upstream inlets of the HYAAC, place the metal tube end previously attached to the syringe containing the cell suspension and load the device. Check loading efficiency, loading more cells if necessary (see Notes 14 and 15).

• 8. Once the device is loaded, remove the metal tube and replace it with a metal pin.

• 9. Set the syringe pump to a speed of 10 μL/min and flow media through it (see Note 16). Monitor the experiment, checking on cell division (Fig. 2E), media levels, and clogging issues (see Note 17).

• 10. Capture images using a microscope with a 40× objective and equipped with an automated stage to allow for multiple X and Y positions to be imaged every 15 min until cells stop dividing (see Note 18).

• 11. The analysis is performed by comparing the sequential images acquired using Fiji software. Doubling time is measured by calculating the time it takes for the mother cell to complete each division. RLS is assessed by counting the total number of offspring produced by each single mother cell. Plot the percentage of viable cells/reproducing cells vs. the generation of daughter cells. Images can also be analyzed to determine cell size, morphological changes, and protein localization.

4 Notes

• 1. Agar media in plates must be originally made thicker; as the microdissection proceeds, the agar will desiccate. It is thus important to use thick-layered agar plates. YPD or synthetic media may be used for microdissection. Additionally, drugs/compounds may be added to the media to evaluate their effect on RLS.

• 2. Each channel enables the analysis of one strain; therefore, this device can quantify the RLS of up to ten different strains simultaneously.

• 3. This design of the microchip, an inverted triangle with angled walls, ensures that daughter cells can be washed off the system, not causing clumps, while allowing trapped mother cells to grow (4–10 μm) and bud in different directions.

• 4. According to the length of the assay, different syringe sizes can be used.

• 5. Tubing, pins, and adaptors must fit well into each other to prevent leakage.

• 6. Using a fluorescent microscope allows the analysis of aged-related phenotypical characteristics, including organelle morphology, gene expression, and protein localization.

• 7. Altering media types and nutrient content of the medium may affect the lifespan of C. neoformans.

• 8. Microdissections are performed at room temperature. It is useful to mark with a pen the area where the selected mother cells are outside the plate as a reference point on the plate. As a visual aid to keep count of the arrayed line, for every five cells, one cell can be kept slightly dislocated to the right of the line.

• 9. The discard site of cells must be at least two fields of view away from the arrayed line so that the cells will not impair the experiment as they keep budding.

• 10. Do not incubate the plate for more than 2 h so as not to generate multiple daughter cells, therefore making it difficult to differentiate the mother cell. As the experiment progresses, the agar medium will shrink, and the microdissection needle will have difficulty reaching the agar. If necessary, holes can be opened in the sides of the plates using hot pliers to facilitate needle movement.

• 11. Cells will replicate at 4 °C over a period of 1–2 days. They should not be kept at 4 °C for a period of over 2 days due to the risk of overgrowth.

• 12. Cells can have a delayed replication cycle; a minimum of 24 h is necessary before considering the cell under senescence. They should be watched for a couple of days to confirm death.

• 13. Using a chemically defined media is suggested to improve reproducibility.

• 14. It is noteworthy that the device is not able to selectively trap only naive cells at the beginning of the experiment. However, in an exponential culture, the majority (~ 80%) of yeast cells are unbudded [8].

• 15. The single-cell trapping efficiency rate should be approximately 60%, and there is a risk of channel clogging and loss of cells over time, especially within the first seven generations. Also, since this platform is in a stable, controlled temperature, it minimizes variations due to environmental temperature switching.

• 16. This constant flow of fresh medium provides continuous nutritional conditions to the cells. It removes daughter cells while the mother cell is trapped in the buckets and can be monitored for the entirety of the experiment. Due to the low flow resistance, only one cell tends to be trapped within an empty bucket. After the trap becomes occupied, there is an increase in the hydrodynamic resistance, ensuring that only single cells are kept in their buckets. By diverting the fluid flow around trapped cells, oncoming cells bypass the occupied traps in preference for empty buckets while also preventing trapped cells from being pulled out of the array. This same system also aids the removal of daughter cells, reducing the physical stress at the cellular level caused by operators manipulating the daughter cells away from each mother cell. If the new bud is oriented upstream of the bucket, the diverted flow removes the daughter cell to the side; if the new bud is oriented downstream, it gets removed between the pillars of the bucket walls [10, 11].

• 17. If the media runs out during the experiment, the syringe pump should be paused to hold the pressure within the system. Load fresh media into a new syringe. Then, remove the metal pin from the inlet connected to the old media-filling syringe. Next, insert the tubing from the new syringe, and fill the new tubing with fresh media while the old syringe is still in place. This approach will hold the pressure within the system, preventing fluid pressure changes that can cause backflow and loss of cells within the device. Place the new syringe and restart the syringe pump. Once pressure has been reestablished, pause the pump to remove the old syringe tubing and reinsert the metal pin.

• 18. During the capture of images, care must be taken during prolonged assays, including overnight experiments, to avoid liquid media spills and leaks in the system that may cause damage to the microscope. Long experiments are more prone to clog and result in overgrowth of cells. Another limitation is providing enough media flowing through the system because the syringes only hold a small volume. Unfortunately, refilling the syringes may disturb the flow, and cells are flushed out of their buckets.