Short-Range C-Signaling Restricts Cheating Behavior during Myxococcus xanthus
Data files
Jun 06, 2022 version files 9.59 GB
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README.rtf
Sep 10, 2024 version files 32.74 GB
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README.md
Sep 16, 2024 version files 32.74 GB
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r3_1_1_NFB1.nd2
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r3_1_2_18_h_100X.nd2
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r3_1_2_24_h_100X_1_fov.nd2
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r3_1_2_24_h_100X_center.nd2
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r3_1_2_24_h_100X_NFB.nd2
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r3_1_2_center1.nd2
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r3_1_2_FOV1.nd2
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r3_1_2_NFB1.nd2
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r3_2_1__NFB1.nd2
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r3_2_1_18_h_100X.nd2
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r3_2_1_24_h_100X_1_fov.nd2
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r3_2_1_24_h_100X_center.nd2
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r3_2_1_24_h_100X_NFB.nd2
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r3_2_1_center1.nd2
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r3_2_1_FOV1.nd2
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r3_4_1_18_h_100X.nd2
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r3_4_1_24_h_100X_1_fov.nd2
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r3_4_1_24_h_100X_center.nd2
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r3_4_1_24_h_100X_NFB.nd2
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r3_4_1_center1.nd2
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r3_4_1_FOV1.nd2
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r3_4_1_NFB1.nd2
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r4_1_1_18_h_100X.nd2
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r4_1_1_24_h_100X_1_fov.nd2
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r4_1_1_24_h_100X_center.nd2
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r4_1_1_24_h_100X_NFB.nd2
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r4_1_1_center.nd2
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r4_1_1_FOV1.nd2
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r4_1_1_NFB.nd2
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r4_1_2_18_h_100X.nd2
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r4_1_2_24_h_100X_1_fov_2.nd2
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r4_1_2_24_h_100X_1_fov.nd2
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r4_1_2_24_h_100X_center.nd2
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r4_1_2_24_h_100X_NFB_2.nd2
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r4_1_2_24_h_100X_NFB.nd2
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r4_1_2_center.nd2
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r4_1_2_FOV1.nd2
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r4_1_2_NFB.nd2
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r4_2_1_18_h_100X.nd2
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r4_2_1_24_h_100X_1_fov.nd2
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r4_2_1_24_h_100X_center.nd2
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r4_2_1_24_h_100X_NFB.nd2
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r4_2_1_center.nd2
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r4_2_1_FOV1.nd2
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r4_2_1_NFB.nd2
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r4_4_1_18_h_100X.nd2
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r4_4_1_24_h_100X_1_fov.nd2
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r4_4_1_24_h_100X_center.nd2
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r4_4_1_24_h_100X_NFB.nd2
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r4_4_1_center.nd2
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r4_4_1_FOV1.nd2
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r4_4_1_FOV2.nd2
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r4_4_1_NFB.nd2
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r5_1_1_18_h_100X.nd2
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r5_1_1_24_h_100X_1_fov.nd2
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r5_1_1_24_h_100X_center.nd2
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r5_1_1_24_h_100X_NFB.nd2
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r5_1_1_center.nd2
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r5_1_1_FOV1.nd2
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r5_1_1_NFB.nd2
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r5_1_2_18_h_100X.nd2
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r5_1_2_24_h_100X_1_fov_2.nd2
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r5_1_2_24_h_100X_1_fov.nd2
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r5_1_2_24_h_100X_center.nd2
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r5_1_2_24_h_100X_NFB_2.nd2
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r5_1_2_24_h_100X_NFB.nd2
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r5_1_2_center.nd2
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r5_1_2_FOV1.nd2
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r5_1_2_NFB.nd2
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r5_2_1_18_h_100X.nd2
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r5_2_1_24_h_100X_1_fov.nd2
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r5_2_1_24_h_100X_center.nd2
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r5_2_1_24_h_100X_NFB.nd2
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r5_2_1_center.nd2
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r5_2_1_FOV1.nd2
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r5_2_1_NFB.nd2
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r5_4_1_18_h_100X.nd2
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r5_4_1_24_h_100X_1_fov.nd2
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r5_4_1_24_h_100X_center.nd2
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r5_4_1_24_h_100X_NFB.nd2
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r5_4_1_center.nd2
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r5_4_1_FOV1.nd2
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r5_4_1_FOV2.nd2
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r5_4_1_NFB.nd2
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r6_1_1_18_h_100X.nd2
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r6_1_1_center.nd2
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r6_1_1_FOV1.nd2
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r6_1_1_NFB.nd2
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r6_1_2_18_h_100X.nd2
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r6_1_2_center.nd2
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r6_1_2_FOV1.nd2
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r6_1_2_NFB.nd2
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r6_2_1_18_h_100X.nd2
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r6_2_1_center.nd2
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r6_2_1_FOV1.nd2
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r6_2_1_NFB.nd2
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r6_4_1_18_h_100X.nd2
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r6_4_1_center.nd2
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r6_4_1_FOV1.nd2
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r6_4_1_FOV2.nd2
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r6_4_1_NFB.nd2
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README.md
Abstract
Myxococcus xanthus uses short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development. A csgA mutant deficient in C-signaling can cheat on wild type (WT) in mixtures and form spores disproportionately, but our understanding of cheating behavior is incomplete. We subjected mixtures of WT and csgA cells at different ratios to co-development and used confocal microscopy and image analysis to quantify the arrangement and morphology of cells. At a ratio of one WT to four csgA cells (1:4), mounds failed to form. At 1:2, only a few mounds and spores formed. At 1:1, mounds formed with a similar number and arrangement of WT and csgA rods early in development, but later the number of csgA spores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT. This cheating after mound formation involved csgA forming spores at a greater rate while WT disappeared at a greater rate, either lysing or exiting NFBs. At 2:1 and 4:1, csgA rods were more abundant than expected throughout the biofilm both before and during mound formation, and cheating continued after mound formation. We conclude that C-signaling restricts cheating behavior by requiring sufficient WT cells in mixtures. Excess cheaters may interfere with positive feedback loops that depend on the cellular arrangement to enhance C-signaling during mound building. Since long-range signaling could not likewise communicate the cellular arrangement, we propose that C-signaling was favored evolutionarily and that other short-range signaling mechanisms provided selective advantages in bacterial biofilm and multicellular animal development.
README: Short-Range C-Signaling Restricts Cheating Behavior during Myxococcus xanthus Development
https://doi.org/10.5061/dryad.tmpg4f51d
Description of the data and file structure
*Growth and development. *M. xanthus was grown and submerged culture development. Briefly, cells were grown at 32°C in CTTYE liquid medium (CTT with 0.2% yeast extract) with shaking at 350 rpm. Development was performed in 8-well μ-slides (Ibidi #80826) with starvation buffer MC7 (10 mM morpholinepropanesulfonic acid [MOPS, pH 7.0], 1 mM CaCl2). Cells from log-phase CTTYE cultures were collected by centrifugation and resuspended to 1,000 Klett units in MC7 containing 0.5 mM vanillate. The cell suspensions were mixed as described in the text and the figure legends. Each cell suspension (30 μL) was then added to 201 μL of MC7 containing 0.5 mM vanillate, and 200 μL was pipeted into each μ-slide well. The μ-slide was wrapped in clear plastic and incubated at 32°C in a humid chamber.
Strains.
M. xanthus** strains** | Description |
---|---|
DK1622 | Laboratory wild-type strain (unlabeled) |
MRR33 | csgA::pRR028 (unlabeled) |
YH7 | DK1622 with MXAN_0018-MXAN_0019::pYH7 (green) |
YH11 | MRR33 with MXAN_0018-MXAN_0019::pYH8 (red) |
Experimental design. For all figures and analyses (except Figure 5 and Figure S12) in the manuscript, half of the WT cells were labeled with the strain YH7 (green fluorescence), while the other half were unlabeled and represented by the strain DK1622. Similarly, for the csgA cells, half were labeled with the strain YH11 (red fluorescence), and the other half were unlabeled and represented by the strain MRR33. The z-stacks are listed in the table below.
ID | Ratio | Hours post-starvation |
---|---|---|
1 | 4:1 | Indicated in the file name |
2 | 4:1 | Indicated in the file name |
3 | 4:1 | Indicated in the file name |
4 | 4:1 | Indicated in the file name |
5 | 4:1 | Indicated in the file name |
6 | 2:1 | Indicated in the file name |
7 | 2:1 | Indicated in the file name |
8 | 2:1 | Indicated in the file name |
9 | 2:1 | Indicated in the file name |
10 | 2:1 | Indicated in the file name |
11 | 1:1 | Indicated in the file name |
12 | 1:1 | Indicated in the file name |
13 | 1:1 | Indicated in the file name |
14 | 1:1 | Indicated in the file name |
15 | 1:1 | Indicated in the file name |
16 | 1:2 | Indicated in the file name |
17 | 1:2 | Indicated in the file name |
18 | 1:2 | Indicated in the file name |
19 | 1:2 | Indicated in the file name |
20 | 1:2 | Indicated in the file name |
For Figures 5 and S12 in the manuscript*,* one-third of the WT cells were labeled with the strain YH7 (green fluorescence), while the remaining two-thirds were unlabeled and represented by the strain DK1622. Similarly, for the csgA cells, one-third were labeled with the strain YH11 (red fluorescence), and the remaining two-thirds were unlabeled and represented by the strain MRR33. The z-stacks are listed in the table below.
Number | Name | Note |
---|---|---|
1 | r1 1_1 18h 100X | Biofilm, 18 h |
2 | r1 1_1 18h 100X_2 | Biofilm, 18 h |
3 | r1 1_2 18h 100X | Biofilm, 18 h |
4 | r1 1_2 18h 100X_2 | Biofilm, 18 h |
5 | r1 2_1 18h 100X | Biofilm, 18 h |
6 | r1 2_1 18h 100X_2 | Biofilm, 18 h |
7 | r1 4_1 18h 100X | Biofilm, 18 h |
8 | r1 4_1 18h 100X_2 | Biofilm, 18 h |
9 | r2 1_1 18h 100X | Biofilm, 18 h |
10 | r2 1_2 18h 100X | Biofilm, 18 h |
11 | r2 2_1 18h 100X | Biofilm, 18 h |
12 | r2 4_1 18h 100X | Biofilm, 18 h |
13 | r3 1_1 18h 100X | Biofilm, 18 h |
14 | r3 1_2 18h 100X | Biofilm, 18 h |
15 | r3 2_1 18h 100X | Biofilm, 18 h |
16 | r3 4_1 18h 100X | Biofilm, 18 h |
17 | r4 1_1 18h 100X | Biofilm, 18 h |
18 | r4 1_2 18h 100X | Biofilm, 18 h |
19 | r4 2_1 18h 100X | Biofilm, 18 h |
20 | r4 4_1 18h 100X | Biofilm, 18 h |
21 | r5 1_1 18h 100X | Biofilm, 18 h |
22 | r5 1_2 18h 100X | Biofilm, 18 h |
23 | r5 2_1 18h 100X | Biofilm, 18 h |
24 | r5 4_1 18h 100X | Biofilm, 18 h |
25 | r6 1_1 18h 100X | Biofilm, 18 h |
26 | r6 1_2 18h 100X | Biofilm, 18 h |
27 | r6 2_1 18h 100X | Biofilm, 18 h |
28 | r6 4_1 18h 100X | Biofilm, 18 h |
29 | r1 1_1 24h 100X NFB | NFB, 24 h |
30 | r1 1_2 24h 100X NFB | NFB, 24 h |
31 | r1 1_2 24h 100X NFB_2 | NFB, 24 h |
32 | r1 2_1 24h 100X NFB | NFB, 24 h |
33 | r1 4_1 24h 100X NFB | NFB, 24 h |
34 | r1 4_1 24h 100X NFB_2 | NFB, 24 h |
35 | r2 1_1 24h 100X NFB | NFB, 24 h |
36 | r2 1_2 24h 100X NFB | NFB, 24 h |
37 | r2 2_1 24h 100X NFB | NFB, 24 h |
38 | r2 4_1 24h 100X NFB | NFB, 24 h |
39 | r3 1_1 24h 100X NFB | NFB, 24 h |
40 | r3 1_2 24h 100X NFB | NFB, 24 h |
41 | r3 2_1 24h 100X NFB | NFB, 24 h |
42 | r3 4_1 24h 100X NFB | NFB, 24 h |
43 | r4 1_1 24h 100X NFB | NFB, 24 h |
44 | r4 1_2 24h 100X NFB | NFB, 24 h |
45 | r4 1_2 24h 100X NFB_2 | NFB, 24 h |
46 | r4 2_1 24h 100X NFB | NFB, 24 h |
47 | r4 4_1 24h 100X NFB | NFB, 24 h |
48 | r5 1_1 24h 100X NFB | NFB, 24 h |
49 | r5 1_2 24h 100X NFB | NFB, 24 h |
50 | r5 1_2 24h 100X NFB_2 | NFB, 24 h |
51 | r5 2_1 24h 100X NFB | NFB, 24 h |
52 | r5 4_1 24h 100X NFB | NFB, 24 h |
53 | r1 1_1 24 h 100X 1 fov | Proximal, 24 h |
54 | r1 1_2 24 h 100X 1 fov | Proximal, 24 h |
55 | r1 2_1 24 h 100X 1 fov | Proximal, 24 h |
56 | r1 4_1 24 h 100X 1 fov | Proximal, 24 h |
57 | r2 1_1 24 h 100X 1 fov | Proximal, 24 h |
58 | r2 1_2 24 h 100X 1 fov | Proximal, 24 h |
59 | r2 2_1 24 h 100X 1 fov | Proximal, 24 h |
60 | r2 4_1 24 h 100X 1 fov | Proximal, 24 h |
61 | r3 1_1 24 h 100X 1 fov | Proximal, 24 h |
62 | r3 1_2 24 h 100X 1 fov | Proximal, 24 h |
63 | r3 2_1 24 h 100X 1 fov | Proximal, 24 h |
64 | r3 4_1 24 h 100X 1 fov | Proximal, 24 h |
65 | r4 1_1 24 h 100X 1 fov | Proximal, 24 h |
66 | r4 1_2 24 h 100X 1 fov | Proximal, 24 h |
67 | r4 1_2 24 h 100X 1 fov_2 | Proximal, 24 h |
68 | r4 2_1 24 h 100X 1 fov | Proximal, 24 h |
69 | r4 4_1 24 h 100X 1 fov | Proximal, 24 h |
70 | r5 1_1 24 h 100X 1 fov | Proximal, 24 h |
71 | r5 1_2 24 h 100X 1 fov | Proximal, 24 h |
72 | r5 1_2 24 h 100X 1 fov_2 | Proximal, 24 h |
73 | r5 2_1 24 h 100X 1 fov | Proximal, 24 h |
74 | r5 4_1 24 h 100X 1 fov | Proximal, 24 h |
75 | r1 1_1 24 h 100X center | Distal, 24 h |
76 | r1 1_2 24 h 100X center | Distal, 24 h |
77 | r1 2_1 24 h 100X center | Distal, 24 h |
78 | r1 4_1 24 h 100X center | Distal, 24 h |
79 | r2 1_1 24 h 100X center | Distal, 24 h |
80 | r2 1_2 24 h 100X center | Distal, 24 h |
81 | r2 2_1 24 h 100X center | Distal, 24 h |
82 | r2 4_1 24 h 100X center | Distal, 24 h |
83 | r3 1_1 24 h 100X center | Distal, 24 h |
84 | r3 1_2 24 h 100X center | Distal, 24 h |
85 | r3 2_1 24 h 100X center | Distal, 24 h |
86 | r3 4_1 24 h 100X center | Distal, 24 h |
87 | r4 1_1 24 h 100X center | Distal, 24 h |
88 | r4 1_2 24 h 100X center | Distal, 24 h |
89 | r4 2_1 24 h 100X center | Distal, 24 h |
90 | r4 4_1 24 h 100X center | Distal, 24 h |
91 | r5 1_1 24 h 100X center | Distal, 24 h |
92 | r5 1_2 24 h 100X center | Distal, 24 h |
93 | r5 2_1 24 h 100X center | Distal, 24 h |
94 | r5 4_1 24 h 100X center | Distal, 24 h |
95 | r1 1_1 NFB1 | NFB, 30 h |
96 | r1 1_2 NFB1 | NFB, 30 h |
97 | r1 2_1 NFB1 | NFB, 30 h |
98 | r1 4_1 NFB1 | NFB, 30 h |
99 | r2 1_1 NFB1 | NFB, 30 h |
100 | r2 1_2 NFB1 | NFB, 30 h |
101 | r2 2_1 NFB1 | NFB, 30 h |
102 | r2 4_1 NFB1 | NFB, 30 h |
103 | r3 1_1 NFB1 | NFB, 30 h |
104 | r3 1_2 NFB1 | NFB, 30 h |
105 | r3 2_1 NFB1 | NFB, 30 h |
106 | r3 4_1 NFB1 | NFB, 30 h |
107 | r4 1_1 NFB1 | NFB, 30 h |
108 | r4 1_2 NFB1 | NFB, 30 h |
109 | r4 2_1 NFB1 | NFB, 30 h |
110 | r4 4_1 NFB1 | NFB, 30 h |
111 | r5 1_1 NFB1 | NFB, 30 h |
112 | r5 1_2 NFB1 | NFB, 30 h |
113 | r5 2_1 NFB1 | NFB, 30 h |
114 | r5 4_1 NFB1 | NFB, 30 h |
115 | r6 1_1 NFB1 | NFB, 30 h |
116 | r6 1_2 NFB1 | NFB, 30 h |
117 | r6 2_1 NFB1 | NFB, 30 h |
118 | r6 4_1 NFB1 | NFB, 30 h |
119 | r1 1_1 FOV1 | Proximal, 30 h |
120 | r1 1_2 FOV1 | Proximal, 30 h |
121 | r1 2_1 FOV1 | Proximal, 30 h |
122 | r1 4_1 FOV1 | Proximal, 30 h |
123 | r2 1_1 FOV1 | Proximal, 30 h |
124 | r2 1_2 FOV1 | Proximal, 30 h |
125 | r2 2_1 FOV1 | Proximal, 30 h |
126 | r2 4_1 FOV1 | Proximal, 30 h |
127 | r3 1_1 FOV1 | Proximal, 30 h |
128 | r3 1_2 FOV1 | Proximal, 30 h |
129 | r3 2_1 FOV1 | Proximal, 30 h |
130 | r3 4_1 FOV1 | Proximal, 30 h |
131 | r4 1_1 FOV1 | Proximal, 30 h |
132 | r4 1_2 FOV1 | Proximal, 30 h |
133 | r4 2_1 FOV1 | Proximal, 30 h |
134 | r4 4_1 FOV1 | Proximal, 30 h |
135 | r4 4_1 FOV2 | Proximal, 30 h |
136 | r5 1_1 FOV1 | Proximal, 30 h |
137 | r5 1_2 FOV1 | Proximal, 30 h |
138 | r5 2_1 FOV1 | Proximal, 30 h |
139 | r5 4_1 FOV1 | Proximal, 30 h |
140 | r5 4_1 FOV2 | Proximal, 30 h |
141 | r6 1_1 FOV1 | Proximal, 30 h |
142 | r6 1_2 FOV1 | Proximal, 30 h |
143 | r6 2_1 FOV1 | Proximal, 30 h |
144 | r6 4_1 FOV1 | Proximal, 30 h |
145 | r6 4_1 FOV2 | Proximal, 30 h |
146 | r1 1_1 center1 | Distal, 30 h |
147 | r1 1_2 center1 | Distal, 30 h |
148 | r1 2_1 center1 | Distal, 30 h |
149 | r1 4_1 center1 | Distal, 30 h |
150 | r2 1_1 center1 | Distal, 30 h |
151 | r2 1_2 center1 | Distal, 30 h |
152 | r2 2_1 center1 | Distal, 30 h |
153 | r2 4_1 center1 | Distal, 30 h |
154 | r3 1_1 center1 | Distal, 30 h |
155 | r3 1_2 center1 | Distal, 30 h |
156 | r3 2_1 center1 | Distal, 30 h |
157 | r3 4_1 center1 | Distal, 30 h |
158 | r4 1_1 center1 | Distal, 30 h |
159 | r4 1_2 center1 | Distal, 30 h |
160 | r4 2_1 center1 | Distal, 30 h |
161 | r4 4_1 center1 | Distal, 30 h |
162 | r5 1_1 center1 | Distal, 30 h |
163 | r5 1_2 center1 | Distal, 30 h |
164 | r5 2_1 center1 | Distal, 30 h |
165 | r5 4_1 center1 | Distal, 30 h |
166 | r6 1_1 center1 | Distal, 30 h |
167 | r6 1_2 center1 | Distal, 30 h |
168 | r6 2_1 center1 | Distal, 30 h |
169 | r6 4_1 center1 | Distal, 30 h |
Code/software
The original raw image data was in .nd2 file, which can be opened with ImageJ, a free open-source tool.
Methods
Images of nascent fruiting bodies were acquired with a Nikon A1 Laser Scanning Confocal Microscope, which was configured on a Nikon Ti inverted platform with an XY automated stage and a 100X objective. Fluorescence from tdTomato were examined using a 560-nm laser for excitation and a 595/50 band pass emission filter. Fluorescence from mNeonGreen was examined using a 488 nm laser for excitation and a 525/50 band pass emission filter. Images near the bottom of NFBs were the first optical section above the bottom of the well, in which cells could be clearly visualized (0.25 to 0.5 μm above the bottom of the well).