Multi-color fluorescence live-cell imaging in Dictyostelium discoideum

Abstract

The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy.Key words: fluorescence imaging, organelle, cytoskeleton, small GTPase, Dictyostelium.

Keywords: Dictyostelium; cytoskeleton; fluorescence imaging; organelle; small GTPase.

Fig. 1

Achilles as a superior green fluorescent protein alternative in D. discoideum (A) Vegetative cells expressing Achilles (upper panels) and mNeonGreen (lower panels) under act15 promoter (left: brightfield channel, right: green channel). Scale bar, 10 μm. (B) Violin plot of the single-cell mean fluorescence intensity distribution of Achilles and mNeonGreen expressing vegetative cells (Achilles, n = 297 cells. mNeonGreen, n = 266 cells). The black line is the median. *: P<10^–15. (C) A slug harboring Achilles (upper panels) and mNeonGreen (lower panel) act15 promoter expression plasmids (left: brightfield channel, right: green channel). Scale bar, 100 μm. (D) High magnification images of slugs harboring Achilles (upper panels) and mNeonGreen (lower panels) act15 promoter expression plasmids (left: brightfield channel, right: green channel). The anterior-posterior axis of the slug is from left to right. Scale bar, 10 μm. (E) Violin plot of fluorescent intensities measured in the anterior and the posterior region of a slug (Achilles, n = 10 slugs. mNeonGreen, n = 10 slugs). The black line indicates the median value.*: P<10^–4. **: P<0.05. NS: not significant (P>0.05).

Fig. 2

Use of Achilles as a yellow fluorescent protein tag in D. discoideum (A) Cells carrying coaAp:PH_Akt-Achilles (upper panels) and coaAp:PH_Akt-mNeonGreen (lower panels) (left: brightfield channel, right: green channel). Scale bar, 10 μm. (B) Violin plot of the single-cell mean fluorescence intensity distribution of PH_Akt-Achilles and PH_Akt-mNeonGreen expressing vegetative cells (PH_Akt-Achilles, n = 402 cells. PH_Akt-mNeonGreen, n = 599 cells). The black line indicates the median. *: P<10^–15. (C) Slugs (PH_Akt-Achilles or -mNeonGreen under coaA promoter. Scale bar, 100 μm. (D) High magnification images of a slug (upper panel PH_Akt-Achilles, lower panels PH_Akt-mNeonGreen). The anterior-posterior axis of the slug is from left to right. Scale bar, 10 μm. (E) Violin plot of the mean fluorescent intensities of the anterior and the posterior region of a slug (PH_Akt-Achilles, n = 12 slugs. PH_Akt-mNeonGreen, n = 12 slugs). The black line indicates the median value.*: P<10^–5. **: P<0.05. ***: P<0.01. NS: not significant (P>0.05).

Fig. 3

Achilles and labile-Achilles as fast responding reporter genes in D. discoideum (A) Dox-induced GFP(S65T) (upper panel) and Achilles (lower panel) fluorescence in vegetative cells plated from shaken culture. Cells were incubated with 10 μg/mL Dox for indicated time (0, 3, 6, and 22 h). WT indicates parental Ax4 cells without expression of fluorescence protein. (B) Percentages of the cells displaying GFP(S65T) or Achilles fluorescence after doxycycline (Dox) addition. Data obtained through flow cytometry (see Material and Methods). Dots and error bars represent the average of positive cells and their standard deviation (three independent experimental runs; n = 135,000 cells, 45,000 cells per experiment). (C) Flow cytometry of V18p:Achilles and V18p:Labile-Achilles. The violin plots represent three independent experimental runs, totaling 90,000 cells (3,000 cells per experiment). The boxes and whiskers indicate the mean values and the interquartile ranges, respectively. Outliers are represented as dots. A Welch Two Sample t-test was conducted to compare the two groups, and significance levels are indicated (***: P<2.2e-16).

Fig. 4

Yellow fluorescent protein Achilles allows early detection of developmental gene expression in D. discoideum (A–C) Streaming-stage cells (8 h after starvation) co-expressing green and red FPs under prestalk and prespore specific-promoter. Panels from left to right: Brightfield images, green fluorescence, red fluorescence and merged image. Scale bars, 100 μm. (A) ecmAOp:Achilles (green) and D19p:mScarlet-I (magenta). (B) ecmAOp:GFP(S65T) (green) and D19p:mScarlet-I (magenta). (C) ecmAOp:Achilles (green) and D19p:mRFPmars (magenta).

Fig. 5

Red fluorescent protein mScarlet-I allows early detection of developmental gene expression in D. discoideum (A–C) Cells co-expressing green and red FPs under prestalk-specific promoter. The numbers indicate hours after nutrient removal. Brightfield images (top panels). Maximum intensity projection of z-stacks (middle and bottom panels). Scale bar, 100 μm. (A) ecmAOp:Achilles (green) and ecmAOp:mRFPmars (magenta). (B) ecmAOp:GFP(S65T) (green) and ecmAOp:mRFPmars (magenta). (C) ecmAOp:Achilles (green) and ecmAOp:mScarlet-I (magenta).

Fig. 6

mScarlet-I is a first-choice red fluorescent protein for protein tagging in D. discoideum (A) act15p:mScarlet-I cells. Vegetative cells (left panels) and cells in a slug (right panels). Brightfield (left panels) and fluorescence images (right panels). Scale bar, 10 μm. (B) act15p:mCherry-RBD_hRaf1 (upper panels) and act15p:mScarlet-I-RBD_hRaf1 (lower panel) expressing vegetative cells. Contrast was adjusted as indicated (top right; color bars). Scale bar, 10 μm. (C) Violin plots of the mean fluorescence intensity of mCherry-RBD_hRaf1 and mScarlet-I-RBD_hRaf1 expressing vegetative cells (mCherry-RBD_hRaf1, n = 298 cells. mScarlet-I-RBD_hRaf1, n = 333 cells). Black line indicates the median. *: P<10^–15. (D) The slug anterior-region of act15p:mCherry-RBD_hRaf1 and act15p:mScarlet-I-RBD_hRaf1 cells. Magnified view (right panels) of the white-boxed area (left panels). The anterior-posterior axis of the slug is from left to right. Scale bar, 100 μm (left) and 10 μm (right). (E) Violin plots of fluorescence intensity distribution in the anterior and the posterior region of the slug expressing mCherry-RBD_hRaf1 or mScarlet-I-RBD_hRaf1 (mCherry-RBD_hRaf1, n = 11 slugs. mScarlet-I-RBD_hRaf1, n = 12 slugs). The black line indicates the median. *: P<10^–5. NS: not significant (P>0.05).

Fig. 7

Blue fluorescent proteins mTurquoise2 and mTagBFP2 for live-cell imaging in D. discoideum (A–H) The brightfield (left panels) and fluorescence images (right panels) of blue fluorescent protein expressing vegetative cells (upper panels) and cells in a slug (lower panels). The anterior-posterior axis of the slug is from left to right. Scale bar, 10 μm. (A) act15p:mTurquoise2 and (B) act15p:mTagBFP2. (C) act15p:mTurquoise2-Lifeact and (D) act15p:mTagBFP2-Lifeact. (E) coaAp:PH_Akt-mTurquoise2 and (F) coaAp:PH_Akt-mTagBFP2. (G) act15p:HistoneH1-mTurquoise2 and (H) act15p:HistoneH1-mTagBFP2.

Fig. 8

Live-cell imaging using large stokes shift LSSmGFP in D. discoideum (A, B) act15p:LSSmGFP and (C, D) control Ax4 cells in the vegetative stage. (A, C) From left to right; brightfield, fluorescent images (405 nm excitation, 447/60 nm emission), (405 nm excitation, 525/50 nm emission), (488 nm excitation, 525/50 nm emission). Scale bars, 10 μm. (B, D) Violin plots of the mean fluorescence intensity of Ax4 (B) or LSSmGFP expressing vegetative cells (D) (Ax4, n = 6 cells. LSSmGFP, n = 6 cells). Black line indicates the median. *: P<0.05. **: P<0.01. NS: not significant (P>0.05). (E–G) Fluorescent images of act15p:LSSmGFP (E), act15p:LSSmGFP-Lifeact (F) and act15p:LSSmGFP-HistoneH1 (G). Vegetative cells (top) and the slug cells (bottom). Brightfield (left panels) and fluorescent images (405 nm excitation, 525/50 nm emission; right panels). The anterior-posterior axis of the slug is left to right. Scale bar, 10 μm. (H) 3-color fluorescence images. (Left to right panels) Brightfield, mitochondria marker act15p:GcvH1(N99)-mTagBFP2, nucleus act15p:LSSmGFP-HistoneH1, plasma membrane coaAp:PKBR1(N150)-Achilles and merged fluorescent channels. Scale bar, 10 μm.

Fig. 9

Live-cell imaging of D. discoideum in near-IR spectrum (A–C) Snapshots of vegetative cells expressing act15p:miRFP670nano3 (A), act15p:HistoneH1-miRFP670nano3 (B) and coaA:PH_Akt-miRFP670nano3 (C). Brightfield (left panels) and fluorescence images (right panels). Scale bars, 10 μm. (D) A slug expressing miRFP670nano3 under the prespore-specific D19 promoter. Brightfield (left), near-IR fluorescence image (middle), merged image (right). Scale bar, 100 μm. (E–G) Snapshots of vegetative cells expressing act15p:Halo (E), act15p:HistoneH1-Halo (F) and coaA:PH_Akt-Halo (G). Left panels, bright field. Right panels NIR fluorescence of Halo-ligand SaraFluor650T. Scale bars, 10 μm. The anterior-posterior axis of the slug is from left to right (A–G).

Fig. 10

4- and 5-color fluorescence live-cell imaging in D. discoideum (A) Vegetative cells expressing 4 FP-tags. From top to bottom: merged channel, act15p:GcvH1(N99)-mTagBFP2 (mitochondria; cyan), coaAp:PKBR1(N150)-Achilles (plasma membrane; yellow), act15p:Dajumin-mScarlet-I (contractile vacuole; magenta), and act15p:HistoneH1-miRFP670nano3 (nuclei; red). White allows: contractile vacuole going through cycles of deformation. Scale bar, 5 μm. (B) Slug cells expressing 5-FP-tags. From top to bottom: merged channel, act15p:GcvH1(N99)-mTagBFP2 (mitochondria; cyan), act15p:LSSmGFP-Lifeact (F-actin; green), act15p:Golvesin-Achilles (Golgi apparatus and ER network; yellow), act5p:PKBR1(N150)-mScarlet-I-2x (plasma membrane; magenta) and act15p:HistoneH1-miRFP670nano3 (nucleus; red). Scale bar, 10 μm.

Fig. 11

Combining cellulose staining with FP-tags to image multicellular body of D. discoideum (A) A migrating slug stained with cellulose-specific dye calcofluor white. Brightfield (top), fluorescence (middle), and merged images (bottom). The slug migrated toward the left side of the images. Scale bar, 100 μm. (B) Time-lapse imaging of culminating D. discoideum stained with cellulose-specific dye Direct Fast Scarlet 4BS (DFS4BS). Brightfield (left), DFS4BS (middle), and merged (right). Slug stage (upper panels) with cellulose-rich trailing slime sheath on agar. A culminant (lower panels) with a stalk tube. Scale bar, 100 μm. (C) Culminating D. discoideum stained with calcofluor white (cyan) expressing Achilles (PstB cell; yellow), mScarlet-I (PstA and PstO cell; magenta), and miRFP670nano3 (prespore cell; red) under ecmB, ecmAO, and D19 promoter, respectively. Scale bar, 100 μm.