Morphological Changes of Mitochondria and Actin Cytoskeleton in the Yeast Saccharomyces cerevisiae During Diauxic Growth and Glucose Depletion Culture

Abstract

We investigated morphological changes of mitochondria and the actin cytoskeleton in the yeast Saccharomyces cerevisiae during diauxic growth and glucose depletion culture. S. cerevisiae exhibits diauxic growth during aerobic culture in a medium containing glucose as a carbon source. We demonstrated that mitochondria in budded cells maintain a tubular morphology at the log phase, but begin to fragment at the diauxic shift phase, in which the metabolism in cells switches from fermentation to respiration due to glucose depletion. On the other hand, the actin cytoskeleton exhibited a polarized distribution in budded cells at the log phase but rapidly depolarized at the diauxic shift phase. The fragmentation of mitochondria and depolarization of the actin cytoskeleton also occurred when cells at the log phase were transferred into a glucose-free medium. In these studies, we found that rapid Dnm1p-dependent fragmentation of mitochondria occurs via glucose depletion. In addition, the results suggest that the changes in mitochondrial morphology and in the polarity of the actin cytoskeleton are independent of each other.

Mitochondria play essential roles in many cellular metabolisms, including the TCA cycle and oxidative phosphorylation. On the other hand, mitochondria maintain their own genome in the nucleoids, and dynamically change their morphology via fusion and fission events (Miyakawa 2017). The yeast Saccharomyces cerevisiae has long been used for studies on mitochondrial morphology. Yeast cells, grown aerobically in a medium containing glucose as a carbon source, maintain tubular mitochondria at the log phase and then form fragmented small mitochondria at the stationary phase. These morphological changes in mitochondria during the growth of yeast cells have been investigated by both electron and fluorescence microscopy (Hoffman and Avers 1973, Stevens 1977, 1981, Sando et al. 1981). These studies revealed that the morphological changes of mitochondria are dynamic and the fragmentation of mitochondria occurs during the growth phase. However, to our knowledge, the exact timing of mitochondrial fragmentation during the growth of yeast cells has not been determined.

S. cerevisiae exhibits diauxic growth in an aerobic culture that contains glucose as a carbon source. Cells rapidly grow during the log phase by fermentation of glucose even in aerobic culture. The growth rate of cells rapidly decreases due to the depletion of glucose at the diauxic shift phase, in which the metabolism switches from fermentation to respiration. Proteomic analysis revealed that many proteins exhibit a significant change in abundance at the diauxic shift phase (Di Bartolomeo et al. 2020). Cells slowly grow during the post-diauxic growth phase and the growth reaches the stationary phase. Based on high-resolution microscopy of yeast cells, the morphology and volume of mitochondria are closely related to the metabolism of cells (Egner et al. 2002). Therefore, the determination of the timing of mitochondrial fragmentation during the growth of yeast cells is important for understanding the correlation of metabolic changes of cells with mitochondrial morphology. In the present study, we focused on the changes in mitochondrial morphology at the diauxic shift phase.

Rhodamine-phalloidin staining revealed that the actin cytoskeleton in yeast cells exhibits two morphologically distinct structures: actin cables and patches (Adams and Pringle 1984). Actin patches are discrete cytoskeletal bodies at the plasma membrane, whereas actin cables are long bundles of actin filaments that are oriented along the mother-bud axis. The motility of mitochondria during the growth of cells depends on actin cables rather than microtubules in S. cerevisiae (Boldogh and Pon 2006, Chernyakov et al. 2013). Therefore, we also investigated the morphological changes in the actin cytoskeleton during the growth of cells. Throughout the experiments, we noted that the glucose concentration in the culture medium is an important factor affecting the morphology of mitochondria. To confirm this assumption, we subsequently investigated the effects of glucose depletion on the morphology of mitochondria and actin cytoskeleton by transferring cells grown in a medium containing glucose into a glucose-free medium. We observed rapid Dnm1p-dependent fragmentation of mitochondria due to glucose depletion. In addition, our study suggests that the changes in mitochondrial morphology and polarity of the actin cytoskeleton are independent of each other.

Materials and methods

Strains and culture

Saccharomyces cerevisiae strains BY4743 (MAT a/α, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, met15Δ0/+, +/lys2Δ0, ura3Δ0/ura3Δ0) and ∆dnm1 (MAT a/α, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, met15Δ0/+, +/lys2Δ0, ura3Δ0/ura3Δ0, dnm1::kanMX4/dnm1::kanMX4) (Giaever et al. 2002) were used. For visualization of mitochondria with GFP, cells were transformed with the pVT100U-mtGFP plasmid (Westermann and Newpert 2000). After preculture on a YPD plate (1% yeast extract, 2% peptone, 2% glucose, and 2% agar), cells were inoculated at OD₆₆₀=0.01 in a Sakaguchi flask that contained 100 mL of YPD medium (1% yeast extract, 2% peptone, and 2% glucose) and cultured aerobically under reciprocal shaking at 30°C. Optical density (OD₆₆₀) was measured in intervals and cells were fixed with 3.7% (w/v) formaldehyde for 1 h at room temperature.

For glucose depletion culture, we used a YP medium (1% yeast extract and 2% peptone) that does not contain glucose. Cells were grown to log phase, harvested by centrifugation at OD₆₆₀=1.0, and washed once with an equal volume of YP medium. Cells were again suspended in an equal volume of YP medium and cultured at 30°C. Cells were collected in intervals and fixed with 3.7% (w/v) formaldehyde for 1 h at room temperature for observation of mitochondria and rhodamine-phalloidin staining.

Fluorescence microscopy

F-actin was visualized using rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR, USA) following the published protocol with slight modifications (Pringle et al. 1989, Miyakawa and Yanagamizu 1998). The formaldehyde-fixed cells were washed twice with SP buffer (0.8 M sorbitol and 25 mM potassium phosphate buffer, pH 7.5), following three washes with solution B (1.2 M sorbitol, 0.5 mM MgCl₂, and 0.1 M potassium phosphate buffer, pH 6.5). Cells were then suspended in PBS (137 mM NaCl, 8.1 mM Na₂HPO₄, 2.68 mM KCl, and 1.47 mM KH₂PO₄) supplemented with 1% Triton X-100 to permeabilize cells. Rhodamine-conjugated phalloidin was added to the cell suspension at a final concentration of 18 U mL⁻¹ and cells were kept in the dark for 30 min at room temperature. The cells were examined using a BHS-RFK or BX53 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan). To measure the percentage of cells, more than 200 cells were counted.

Results

The growth curve of cells that were cultured in the YPD medium is shown in Fig. 1A. Cells grew exponentially during 14 h after the onset of the culture, and the growth rate decreased between 14 h and 16 h. We defined the growth phase between 14 h and 16 h, as the diauxic shift phase. Cells grew with a slower growth rate during the post-diauxic shift phase than during the log phase, and the growth finally reached a stationary phase at 30 h (data not shown). We observed the morphology of mitochondria and actin cytoskeleton in detail between 10 h and 20 h of culture.

Fig. 1. Morphological changes in mitochondria and the actin cytoskeleton during the diauxic growth of S. cerevisiae. (A) Growth curve of BY4743 strain in YPD medium. The optical density of the culture was measured at 660 nm. (B) Classification of cells by the morphology of mitochondria and actin cytoskeleton. According to the morphology of mitochondria, cells were classified into three types: Type 1 cells have tubular mitochondrial networks. Type 2 cells have tubular but partially fragmented mitochondria. Type 3 cells have many small spherical mitochondria. According to the morphology of the actin cytoskeleton, cells were also classified into three types: Type A cells have actin patches highly concentrated in the bud and actin cables elongating in mother cells. Type B cells have actin patches highly concentrated in the bud, but actin patches rather than actin cables are visible in mother cells. Type C cells have actin patches distributed uniformly in both mother and bud cells. Scale bar=5 µm. (C) Changes in mitochondrial morphology in budded cells between 10 and 20 h of culture. Cells were classified into Type 1 (○), Type 2 (□), and Type 3 (△), as shown in Fig. 1B. (D) Changes in actin cytoskeleton in budded cells between 10 and 20 h of culture. Cells were classified into Type A (○), Type B (□), and Type C (△), as shown in Fig. 1B.

The morphology of mitochondria changes during the cell cycle (Sando et al. 1981). The actin cytoskeleton also changes the distribution during the cell cycle (Adams and Pringle 1984). Therefore, to demonstrate that the changes in mitochondria and actin cytoskeleton also depend on the growth phase of cells, it is important to observe cells at the same stage of the cell cycle in each growth phase. In the present study, we observed only cells with small and medium buds, in which the long axis of the bud is less than half of that of the mother cell.

Cells were classified into three types according to the mitochondrial morphology (Fig. 1B). Type 1 cells have networks of tubular mitochondria. Type 2 cells have tubular but partially fragmented mitochondria. Type 3 cells have small fragmented or spherical mitochondria that are distributed in the cytoplasm. According to the morphology of the actin cytoskeleton, cells were also classified into three types. Type A cells have actin patches concentrated in a bud and actin cables elongating in the mother cell. Type B cells have actin patches concentrated in a bud, but actin patches rather than actin cables in the mother cell. Type C cells have actin patches with a random distribution in a bud and the mother cell. Based on these classifications, morphological changes of mitochondria and actin cytoskeleton were observed (Fig. 1C, D). More than 80% of cells belonged to type 1 cell that had tubular mitochondria elongating from mother cells to the bud during the log phase (Fig. 1C). The dominance of type 1 cells continued until cells reached the midpoint of the diauxic shift phase at 15 h. However, the increase in type 2 cells began at 16 h. The increase in type 2 cells suggested that fragmentation of mitochondria gradually continued during the post-diauxic growth phase. Lastly, type 3 cells with small fragmented or spherical mitochondria replaced type 2 cells and occupied more than 70% of cells at 20 h of culture (Fig. 1C).

Through the log phase, more than 90% of cells had a polarized actin cytoskeleton (type A and type B) (Fig. 1D). However, actin depolarization occurred rapidly during the diauxic shift phase and almost all cells transiently changed to type C at 16 h of culture. Actin depolarization persisted for 1 h and cells with a polarized actin cytoskeleton gradually increased after 17 h of culture through the post-diauxic growth phase.

We hypothesized that the fragmentation of mitochondria and depolarization of the actin cytoskeleton was induced by glucose depletion at the diauxic shift phase. To confirm this assumption, we observed the morphology of mitochondria and actin cytoskeleton in cells that were transferred from YPD medium to YP medium that does not contain glucose. Morphological changes in mitochondria and the actin cytoskeleton after glucose depletion are shown in Fig. 2. The mitochondrial morphology and actin cytoskeleton were classified as defined in Fig. 1B. Short-term changes in mitochondria until 40 min of culture are shown in Fig. 2A and the long-term changes until 5 h of culture are shown in Fig. 2B. Short-term changes in the actin cytoskeleton until 40 min of culture are shown in Fig. 2C and the long-term changes until 5 h of culture are shown in Fig. 2D. Representative images of cells are shown in Fig. 2E. More than 90% of cells had tubular mitochondria (type 1 cells) at the time of transfer at 0 h (Fig. 2A, E). However, mitochondrial fragmentation occurred immediately after the transfer of cells to the YP medium, and approximately 90% of cells converted to type 2 cells with fragmented mitochondria after 5–10 min of glucose depletion (Fig. 2A, E). The percentage of type 1 cells with tubular mitochondria began to increase at 15 min after glucose depletion and recovered to 80% after 1 h of glucose depletion culture (Fig. 2B, E). Throughout glucose depletion culture, type 3 cells that harbored small fragmented or spherical mitochondria did not appear. The increase in cell number was negligible during 5 h of culture in the absence of glucose (data not shown).

Fig. 2. Morphological changes in mitochondria and the actin cytoskeleton in wild-type cells during glucose depletion culture. Cells grown to log phase in YPD medium were transferred to glucose-free YP medium. (A, B) Percentage of cells with different mitochondrial morphology during 40 min (A) and 5 h (B) after the onset of glucose depletion culture. Cells were classified into Type 1 (○), Type 2 (□), and Type 3 (△) according to the morphology of mitochondria, as shown in Fig. 1B. (C, D) Percentage of cells with different morphology of the actin cytoskeleton during 40 min (C) and 5 h (D) after the onset of glucose depletion culture. Cells were classified into Type A (○), Type B (□), and Type C (△) according to the morphology of the actin cytoskeleton, as shown in Fig. 1B. (E) Representative images of mitochondria and the actin cytoskeleton during glucose depletion culture. Scale bar=5 µm.

Polarization of the actin cytoskeleton also remarkably changed (Fig. 2C, D, E). More than 90% of cells had a polarized actin cytoskeleton (type A and type B cells) at the time of transfer at 0 h (Fig. 2C, E). However, the polarized distribution of actin patches was rapidly lost 5–10 min after glucose depletion and the state of depolarization of the actin cytoskeleton persisted for 3 h (Fig. 2D, E). The gradual recovery of polarization of the actin cytoskeleton began after 3 h of culture.

Division of mitochondria is driven by dynamin-related GTPase Dnm1p (Otsuga et al. 1998). To determine whether the fragmentation of mitochondria by glucose depletion is dependent on Dnm1p activity, we performed a glucose depletion culture using ∆dnm1 cells (Fig. 3). Mitochondria do not divide into ∆dnm1 cells and all ∆dnm1 cells have tubular mitochondria during culture. Short-term changes in mitochondria and the actin cytoskeleton until 40 min of culture are shown in Fig. 3A and Fig. 3C, respectively. The long-term changes until 5 h of culture are shown in Fig. 3B and Fig. 3D. The morphology of tubular mitochondria in ∆dnm1 cells did not change after glucose depletion culture (Fig. 3A, B, E). On the other hand, rapid depolarization of the actin cytoskeleton and the gradual recovery of actin polarization occurred in ∆dnm1 cells as observed in wild-type cells (Fig. 3C, D, E).

Fig. 3. Morphological changes in mitochondria and the actin cytoskeleton in ∆dnm1 cells during glucose depletion culture. Cells grown to log phase in YPD medium were transferred to glucose-free YP medium, as shown in Fig. 2. (A, B) Percentage of cells with different mitochondrial morphology during 40 min (A) and 5 h (B) after glucose depletion culture. Cells were classified into Type 1 (○), Type 2 (□), and Type 3 (△) according to the morphology of mitochondria, as shown in Fig. 1B. (C, D) Percentage of cells with different morphology of the actin cytoskeleton during 40 min (C) and 5 h (D) after glucose depletion culture. Cells were classified into Type A (○), Type B (□), and Type C (△) according to the morphology of the actin cytoskeleton, as shown in Fig. 1B. (E) Representative images of mitochondria and the actin cytoskeleton during glucose depletion culture. Scale bar=5 µm.

Discussion

In the present study, we revealed that mitochondrial fragmentation in S. cerevisiae cells grown in the YPD medium begins at the diauxic shift phase. As the metabolism in S. cerevisiae cells switches from fermentation to respiration at the diauxic shift phase due to the depletion of glucose, we hypothesized that glucose depletion induces the fragmentation of mitochondria. To assess the effects of glucose depletion, we performed a glucose depletion culture by transferring cells from the YPD medium to the YP medium and demonstrated that Dnm1p-dependent rapid fragmentation of mitochondria is induced by glucose depletion. This strongly suggests the presence of signals that promote mitochondrial fragmentation by sensing the depletion of glucose.

Recently, Zheng et al. (2019) demonstrated that mitochondria in the fission yeast Schizosaccharomyces pombe fragment in a dynamin GTPase Dnm1-dependent manner by glucose starvation. They reported that protein kinase A (PKA) activity is involved in the regulation of the fragmentation of mitochondria and low PKA activity promotes mitochondrial fragmentation (Zheng et al. 2019). In mammalian cells, the fission of mitochondria depends on Drp1, a mammalian homolog of Dnm1. PKA phosphorylates Drp1 at Ser-637 and the phosphorylation of Drp1 inhibits mitochondrial fission by preventing Drp1 from localizing to mitochondria (Chang and Blackstone 2007, Cereghetti et al. 2008). However, the phosphorylation site corresponding to Ser-637 is not conserved in Dnm1 of S. pombe, and the mechanisms by which PKA regulates fragmentation of mitochondria remain unclear.

Ser-637 is not conserved in Dnm1p of S. cerevisiae, similar to S. pombe; therefore, whether PKA is involved in mitochondrial fission in S. cerevisiae is unknown. It should be noted that the response of mitochondrial morphology to glucose depletion differs between S. pombe and S. cerevisiae. The fragmentation of mitochondria in S. pombe began a few minutes after glucose depletion and proceeded for 40 min (Zheng et al. 2019) On the other hand, the fragmentation of mitochondria in S. cerevisiae began within a few minutes, with a maximum peak at 10 min, but the morphology of tubular mitochondria recovered to the original level after 1 h of culture in glucose-free medium (Fig. 2) Intracellular ATP levels decreased by more than 80% within 2 min in S. cerevisiae cells abruptly depleted of glucose, but the ATP level gradually returned to 50% of the original level after 15 min of culture (Xu and Bretscher 2014). The timing of the rapid decrease and restoration of ATP level resembled that of the fragmentation of mitochondria and restoration of tubular mitochondria in our study. It remains to be determined whether the ATP level and mitochondrial morphology are related.

In the present study, we revealed that depolarization of the actin cytoskeleton takes place at the diauxic shift phase. On the other hand, rapid depolarization of the actin cytoskeleton was previously noted in S. cerevisiae by glucose depletion culture (Novick et al. 1989). Uesono et al. (2004) investigated this phenomenon in detail and found that the transient depolarization of actin cytoskeleton rapidly took place within 30 min after transfer of cells from the YPD medium to the YP medium. The polarization of the actin cytoskeleton again recovered slowly after another 5 h of culture. Our study is consistent with that by Uesono et al. (2004). They suggested that the phenomenon induced by glucose depletion contains two pathways: Rapid depolarization of the actin cytoskeleton is dependent on type 1 protein phosphatase Glc7p (Reg1p) and PKA, which does not depend on gene expression. On the other hand, the recovery of actin polarization during 5 h of culture is dependent on respiration activity, Snf1p protein kinase, and gene expression that depends on transcription factors Msn1p and Msn4p (Uesono et al. 2004). Whether depolarization of the actin cytoskeleton at the diauxic shift phase occurs through the mechanisms described above remains to be examined.

As shown in Fig. 3, the tubular morphology of mitochondria did not change despite marked depolarization of the actin cytoskeleton in ∆dnm1 cells. Thus, there is no direct relationship between depolarization of the actin cytoskeleton and mitochondrial fragmentation. As shown in Fig. 1, the changes in the actin cytoskeleton during the diauxic shift phase were more rapid than those in mitochondrial morphology. In the glucose depletion culture, the timing of recovery of tubular mitochondria was faster than that of the appearance of the polarized actin cytoskeleton (Fig. 2). This suggests that the changes in mitochondrial morphology and in the polarity of the actin cytoskeleton independently occur. We consider glucose depletion culture a useful means to investigate the regulatory mechanisms of mitochondria fission in S. cerevisiae.

Acknowledgments

This work was supported by Super Suishinntai from Yamaguchi University.

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