Lipid droplet dynamics during Schizosaccharomyces pombe sporulation and their role in spore survival

Abstract

Upon nitrogen starvation, the fission yeast Schizosaccharomyces pombe forms dormant spores; however, the mechanisms by which a spore sustains life without access to exogenous nutrients remain unclear. Lipid droplets are reservoirs of neutral lipids that act as important cellular energy resources. Using live-cell imaging analysis, we found that the lipid droplets of mother cells redistribute to their nascent spores. Notably, this process was actin polymerization-dependent and facilitated by the leading edge proteins of the forespore membrane. Spores lacking triacylglycerol synthesis, which is essential for lipid droplet formation, failed to germinate. Our results suggest that the lipid droplets are important for the sustenance of life in spores.

Keywords: Actin; Forespore membrane; Germination; Lipid droplet; Septation initiation network; Spore.

Fig. 1.

Dynamics of LDs during spore formation. (A) Representative time-lapse images of the FSMs engulfing the LDs observed in living S. pombe cells expressing the FSM marker mCherry-Psy1 and the LD marker Ptl2-GFP (11 cells observed). FSM and LDs are shown in red and green, respectively. LDs appeared as distinct focal structures. FSM initiation (detected as aggregation of the mCherry-Psy1 fluorescence signals in the cytoplasm) was designated as 0 min. The arrows at 0 min indicate clustering of LDs near the FSM initiation site. The arrowheads at 12 min indicate four LD clusters near the FSM leading edges. The arrowheads at 24 min indicate inclusion of the LDs by FSM extension. Scale bar: 5 µm. (B) Fluorescence images of FSM extension. Cells expressing mCherry-Psy1 were fixed for EM imaging (see Materials and Methods). The numbers 1–4 represent the four FSMs. Scale bar: 2 µm. (C) TEM image of the same cell shown in (B). Scale bar: 2 µm. (D) Magnified TEM images of the cell depicted in (C). Each of the numbered images corresponds to the numbered FSMs in (B). The red arrowheads indicate the FSM. The cyan arrows indicate LDs, which appear as white matter when visualized by TEM. N, nucleus; M, mitochondria. Scale bar: 500 nm. (E) Localization of the LEP rings at the FSM leading edge. N, nucleus. (F-H) Co-localization of LDs and LEPs. Immediately before imaging, the fluorescent dye BODIPY493/503 or BODIPY TR was added to the sporulation medium containing cells expressing Mcp4-mCherry, Meu14-GFP, or LifeAct-GFP. The BODIPY dyes stain LDs. LifeAct-GFP binds to actin filaments to allow visualization of the meiotic actin ring (Yan and Balasubramanian, 2012). Scale bar: 5 µm.

Fig. 2.

Actin polymerization is required for LD clustering. Time-lapse images of living cells expressing mCherry-Psy1 (red) and Ptl2-GFP (green): wild-type cell treated with DMSO (A), wild-type cell treated with Latrunculin A (final concentration of 1 µM) (B), and the mcp4Δ mutant (C). The arrowheads at 24 min indicate the FSM with few associated LDs. The arrows at 48 min represent LDs in the exterior of the forespores. The presented image is a representative example: the number of cells observed is 10 for (A), 12 for (B) and 15 for (C). Scale bar: 5 µm.

Fig. 3.

LEPs are important for efficient inclusion of LDs. (A,B) Representative time-lapse images of LD movements during FSM assembly in the meu14Δ mutant (A) (12 cells observed) and the mug27Δ mutant (B) (14 cells observed); Ptl2-GFP (LDs) and mCherry-Psy1 (FSMs) are shown in green and red, respectively. The arrowheads at 12 min indicate the FSM leading edges, without association of the LDs in the meu14Δ mutant. The arrows at 48 min indicate the LDs remaining in the ascus cytoplasm. Scale bar: 5 µm. (C) Quantification of LDs enclosed by FSMs in the various strains. Time-lapse images of 10 samples for each strain were counted. The number of LDs in a sporulating cell was quantified at 48 min or after. Percentage of LDs transported into the forespore=(number of LDs enclosed by the FSM/total number of LDs)×100%. The graph and the error bar represent mean and standard deviation, respectively.

Fig. 4.

LDs are important for spore germination and spore wall integrity. (A) Frequency of tetrad formation and spore germination in the different strains. Tetrad formation frequency=(number of asci with four spores/number of total zygotes)×100%. At least 600 tetrads were scored for each strain. After 3-5 days of sporulation, the rate of spore germination of each strain was determined by assessing spore colony formation through tetrad analysis (42 tetrads were dissected for each strain). Spores that failed to form colonies were further confirmed for germination using a dissecting microscope. Germination frequency=(number of germinated spores/number of total spores)×100%. A lys1⁺-integrating plasmid carrying dga1⁺ or plh1⁺ was used to restore the germination efficiency of the dga1Δplh1Δ mutant. (B) The dga1Δplh1Δ mutant possessed few LDs. The fluorescent dye BODIPY was used for LD labeling. The white dashed line outlines the sporulating cell Scale bar: 5 µm. (C) Examples of spore colony formation in the dga1Δplh1Δ mutant. (D) Representative morphological changes of spore germination over time in the wild-type or the dga1Δplh1Δ spore. Scale bar: 6 µm. (E) The germination defect of the dga1Δplh1Δ spores was time-dependent. The cells were subjected to sporulation on an ME plate for 2, 4, 8, or 16 days. At each time point, spore germination was assayed by tetrad dissection on the YES plate (three independent experiments per strain; 14 tetrads were dissected per experiment). Germination frequency=(number of germinated spores/number of total spores)×100%. The graph and the error bar represent mean and standard deviation, respectively. (F) The spore wall was improperly assembled in the dga1Δplh1Δ mutant. Isp3-GFP was used to visualize the outermost layer of the spore wall. Isp3-GFP fluorescence signals were evenly distributed on the surface of the wild-type ascospores, whereas Isp3-GFP exhibited aggregate formation and uneven decoration of the ascospores of the dga1Δplh1Δ mutant. Scale bar: 5 µm.