Copine A plays a role in the differentiation of stalk cells and the initiation of culmination in Dictyostelium development

Abstract

Background: Copines are calcium-dependent phospholipid-binding proteins found in diverse eukaryotic organisms. We are studying the function of copines in Dictyostelium discoideum, a single-celled amoeba that undergoes cell differentiation and morphogenesis to form multicellular fruiting bodies when placed in starvation conditions. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to complete the developmental cycle, arresting at the slug stage. The aim of this study is to further characterize the developmental defect of the cpnA- cells.

Results: Time-lapse imaging revealed that cpnA- cells exhibited delayed aggregation and made large mounds that formed one large slug as compared to the smaller slugs of the wild-type cells. While the prespore cell patterning appeared to be normal within the cpnA- slugs, the prestalk cell patterning was different from wild-type. When cpnA- cells were mixed with a small percentage of wild-type cells, chimeric fruiting bodies with short stalks formed. When a small percentage of cpnA- cells was mixed with wild-type cells, the cpnA- cells labeled with GFP were found located throughout the chimeric slug and in both the stalk and sporehead of the fruiting bodies. However, there appeared to be a small bias towards cpnA- cells becoming spore cells. When cpnA- cells were developed in buffer containing EGTA, they were also able to differentiate into either stalk or spore cells to form fruiting bodies with short stalks.

Conclusions: Our results indicate that CpnA is involved in the regulation of aggregation, slug size, and culmination during Dictyostelium development. More specifically, CpnA appears to be involved in the function and differentiation of prestalk cells and plays a role in a calcium-regulated signaling pathway critical to triggering the initiation of culmination.

Figure 1

cpnA- cells are arrested in the slug stage of development. Wild-type (A) and cpnA- (C) Dictyostelium cells were plated on black filters at 5 × 10⁷ cells/mL in starvation buffer and developed for 48 hours. Images were taken using a Leica dissecting microscope at 40× magnification (scale bar = 0.5 mm). Wild-type (B) and cpnA- (D) stalk structures were removed from the filters, placed in glass bottom dishes, and imaged using a Nikon fluorescence microscope at 1000× (B) and 600× (D) magnification with DIC optics (scale bar = 40 μm).

Figure 2

Time-lapse imaging revealed that cpnA- cells are delayed in aggregation and form larger mounds that develop into fewer, larger slugs. Wild-type (A-C, G-I) and cpnA- (D-F, J-L) Dictyostelium cells expressing GFP were plated on black filters in starvation buffer at 5 × 10⁷ cells/mL and imaged every 10 minutes for 28 hours using a Leica dissecting fluorescence microscope at 20× magnification (scale bar = 1 mm). Hours:minutes in development are given in the lower right corner of each image. Arrow (K) points to stalk-like structure. Also see time-lapse movies in Additional Files 1 and 2.

Figure 3

Prestalk and prespore cell patterning in cpnA- slugs. Wild-type (A-C) and cpnA- (D-F) Dictyostelium cells transformed with the pspA-Gal (A, D) ecmAO-Gal (B, E), and ecmB-Gal (C, F) plasmids were plated on white filters in starvation buffer at 5 × 10⁷ cells/mL and allowed to develop for 24 hours. At ~16 hours of development, slug structures were fixed and stained with X-Gal to reveal prestalk and prespore patterning. Images were taken with the Nikon SMZ1500 dissecting microscope equipped with a SPOT Flex colored camera using the SPOT digital imaging software (scale bar = 500 μm).

Figure 4

Mixing with 10% wild-type cells begins to rescue the culmination defect of cpnA- cells. Wild-type and cpnA- Dictyostelium cells were mixed at various ratios, plated on black filters in starvation buffer at 5 × 10⁷ cells/mL, and allowed to develop for 24 hours. The wild-type cell percentage is given in the lower right of each image. Images were taken using a Leica dissecting microscope at 20× magnification (scale bar = 1 mm). Arrows (C-F) point to stalks.

Figure 5

Wild-type cells differentiate into stalk or spore cells when mixed with cpnA- cells. Wild-type cells expressing GFP (10%) were mixed with cpnA- cells (90%), plated on black filters in starvation buffer at 5 × 10⁷ cells/mL, and allowed to develop for 24 hours. Fluorescent images of chimeric slugs (A) and fruiting bodies (B) were taken with a Leica dissecting microscope at 50× magnification (scale bar = 0.5 mm). (C) Wild-type cells expressing GFP and cpnA- cells were mixed at various ratios and allowed to develop. Fruiting bodies were removed from the filter, placed on glass bottom dishes, and imaged with a Nikon fluorescence microscope. The percentage of fluorescent spores from each ratio was calculated five times and averaged. Error bars indicate standard error.

Figure 6

cpnA- cell are able to differentiate into either stalk or spore cells when mixed with wild-type cells. cpnA- cells expressing GFP (10%) were mixed with wild-type cells (90%), plated on black filters in starvation buffer at 5 × 10⁷ cells/mL, and allowed to develop for 24 hours. Fluorescent images of chimeric slugs (A) and fruiting bodies (B) were taken with a Leica dissecting microscope at 50× magnification (scale bar = 0.5 mm). (C) cpnA- cells expressing GFP and wild-type cells were mixed at various ratios and allowed to develop. Fruiting bodies were removed from the filter, placed on glass bottom dishes, and imaged with a Nikon fluorescence microscope. The percentage of fluorescent spores from each ratio was calculated five times and averaged. Error bars indicate standard error.

Figure 7

cpnA- cells can differentiate into morphologically normal stalk and spore cells. cpnA- cells expressing GFP (5%) and wild-type cells (95%) were mixed, plated on black filters in starvation buffer at 5 × 10⁷ cells/mL, and allowed to develop for 24 hours. Fruiting bodies were removed from the filter and placed in glass bottom dishes. Spores (A, C) and stalks (B, D) were imaged with DIC (A, B) and fluorescence (C, D) microscopy using a Nikon fluorescence microscope at 600× magnification (scale bar = 40 μm).

Figure 8

In reduced calcium conditions, cpnA- cells form fruiting bodies with short stalks. Wild-type (A, B) and cpnA- (C, D) cells were plated on black filters in starvation buffer with either 2 mM calcium (A, C) or 2 mM EGTA (B, D) at 5 × 10⁷ cells/mL, and allowed to develop for 48 hours. Images were taken using a Leica dissecting microscope at 40× magnification (scale bar = 0.5 mm). Insets are side views of fruiting bodies to show stalk length at the same magnification.

Figure 9

cpnA- cells can differentiate into morphologically normal spore and stalk cells in reduced calcium conditions. Wild-type (A, C) and cpnA- (B, D) cells were allowed to develop in starvation buffer containing EGTA for 48 hours. Fruiting bodies were removed from filters, placed on glass bottom dishes, and imaged with a Nikon microscope at 1000× magnification (scale bar = 20 μm).