Multi-color fluorescence live-cell imaging in Dictyostelium discoideum
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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.
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Summary and Discussion
This is some really neat work. This is a wonderful resource for the community! I really appreciated the logic and decision making highlighted throughout the paper leading to an optimal protocol for 4- or 5- color imaging in dicty! Very cool work and beautiful images!!!
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We constructed strains expressing Achilles or GFP(S65T) under the control of a prestalk-specific ecmAO promoter
Why did y'all shift from mNeonGreen comparisons to GFP?
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22 h was required for a similar percentage of Dox-GFP(S65T)
Is there any data comparing Dox-GFP with a Dox-mNeonGreen FP? If so, are the two probes comparable in their response times?
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These result suggests that despite the low temperature (22 °C) of D. discoideum culture, maturation of Achilles was fast and comparable to that demonstrated in mice
Very neat & exciting finding!!!
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Using the same microscopy setup as above, in vegetative cells, PHAkt-Achilles and PHAkt-mNeonGreen fluorescence appeared localized in the pinocytic cups (Fig. 2A),
Was the cytoplasmic background also brighter here (not just the localized signal)? I'm just naively wondering if the achilles tag is causing some stress that is increasing background autofluorescence in the cell (given the onset of uniform cytoplasmic fluorescence in Fig 1).
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In the slug stage, Achilles fluorescence remained bright in the majority of cells and appeared uniform in the cytosol (Fig. 1C and D), whereas very few cells showed mNeonGreen fluorescence (Fig. 1C–E).
Interesting that there is a significant difference in the distribution of mNeonGreen (anterior vs posterior) but not with Achilles. Do you have any ideas why this would be the case? Are there any observable physiological defects caused by either of these FPs?
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Although various FP has been utilized in D. discoideum (Table S1
Thank you for compiling this list! It is a very useful resource!
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