Using Live-Cell Imaging and Synthetic Biology to Probe Directed Migration in Dictyostelium

Kuhn, Jonathan; Lin, Yiyan; Devreotes, Peter N.

doi:10.3389/fcell.2021.740205

Cited by 11 publications

(12 citation statements)

References 102 publications

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“…This may be of particular interest for applications that require coordinated stoichiometric expression. For instance, two-part optogenetic systems, such as those based on CRY2/CIBN interactions, are of interest in D. discoideum [ 52 ]. These systems have benefited from concerted expression of the two interacting proteins using a 2A peptide in other model organisms [ 9 ].…”

Section: Discussionmentioning

confidence: 99%

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

Zhu

Ricci-Tam²,

Hager³

et al. 2023

PLoS ONE

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The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.

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Section: Discussionmentioning

confidence: 99%

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

Zhu

Ricci-Tam²,

Hager³

et al. 2023

PLoS ONE

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“…For example, cell migration is an important physiological process that occurs during wound healing, embryonic development, and disease (e.g., cancer metastasis). D. discoideum is an ideal model system for studying fundamental aspects of cellular migration, particularly the mechanisms underlying chemotaxis ( Kamimura and Ueda, 2021 ; Kuhn et al, 2021 ; Xu et al, 2021 ). As discussed in this review, neddylation regulates the migration of a variety of cell types ( Park et al, 2018 ; Kim et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum

2022

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“Cullins (CULs) are a core components” of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum, has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum, which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.

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“…All images of Dictyostelium or HL-60 cells were acquired with 63X/1.40 PlanApo oil or 40X/1.30 PlanNeofluar oil DIC objective, respectively, along with digital zoom. In single Dictyostelium or HL-60 cells, confocal imaging was performed at a middle plane of the cells, whereas in giant Dictyostelium cells, laser was focused near the bottom surface of cells to visualize cortical wave propagation 4,35,88,90 . For inhibitor experiments, differentiated HL-60 cells were treated with 20 µM AS605240, 20 µM PP242, 50 µM LY294002, 50 µM CK-666, 50 µM blebbistatin, or 10 µM Y27632 for atleast 10 mins before imaging.…”

Section: Methodsmentioning

confidence: 99%

“…Time interval of photoactivation and image acquisition was ∼10 secs. For imaging HL-60 cells, both microscopes were equipped with a temperature-controlled chamber set at 5% CO 2 and 37 0 C 4,35,88,90 .…”

Section: Methodsmentioning

confidence: 99%

Ras-mediated homeostatic control of front-back signaling dictates cell polarity

Lin,

Pal,

Banerjee

et al. 2023

Preprint

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Studies in the model systems, Dictyostelium amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation on the membrane is spatiotemporally regulated by its activators, RasGEFs, and inhibitors, RasGAPs, which might be expected to create a stable front and back, respectively, in migrating cells. Focusing on C2GAPB in amoebae and RASAL3 in neutrophils, we investigated how Ras activity along the cortex controls polarity. Since existing gene knockout and overexpression studies can be circumvented, we chose optogenetic approaches to assess the immediate, local effects of these Ras regulators on the cell cortex. In both cellular systems, optically targeting the respective RasGAPs to the cell front extinguished existing protrusions and changed the direction of migration, as might be expected. However, when the expression of C2GAPB was induced globally, amoebae polarized within hours. Furthermore, within minutes of globally recruiting either C2GAPB in amoebae or RASAL3 in neutrophils, each cell type polarized and moved more rapidly. Targeting the RasGAPs to the cell backs exaggerated these effects on migration and polarity. Overall, in both cell types, RasGAP-mediated polarization was brought about by increased actomyosin contractility at the back and sustained, localized F-actin polymerization at the front. These experimental results were accurately captured by computational simulations in which Ras levels control front and back feedback loops. The discovery that context-dependent Ras activity on the cell cortex has counterintuitive, unanticipated effects on cell polarity can have important implications for future drug-design strategies targeting oncogenic Ras.

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Using Live-Cell Imaging and Synthetic Biology to Probe Directed Migration in Dictyostelium

Cited by 11 publications

References 102 publications

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum

Ras-mediated homeostatic control of front-back signaling dictates cell polarity

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