-
Scientific Data Jun 2024Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition...
Dicytostelium firmibasis is a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni- to multicellular life. The D. firmibasis genome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-level D. firmibasis genome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison to Dictyostelium discoideum, the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.
Topics: Dictyostelium; Genome, Protozoan; Chromosomes; Molecular Sequence Annotation
PubMed: 38909042
DOI: 10.1038/s41597-024-03513-8 -
The Journal of Cell Biology Sep 2024Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of...
Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.
Topics: Dictyostelium; Pinocytosis; Protozoan Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction
PubMed: 38888895
DOI: 10.1083/jcb.202401110 -
Frontiers in Physiology 2024Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act...
Though myosins share a structurally conserved motor domain, single amino acid variations of active site elements, including the P-loop, switch-1 and switch-2, which act as nucleotide sensors, can substantially determine the kinetic signature of a myosin, ., to either perform fast movement or enable long-range transport and tension generation. Switch-2 essentially contributes to the ATP hydrolysis reaction and determines product release. With few exceptions, class-1 myosin harbor a tyrosine in the switch-2 consensus sequence DIYGFE, at a position where class-2 myosins and a selection of myosins from other classes have a substitution. Here, we addressed the role of the tyrosine in switch-2 of class-1 myosins as potential determinant of the duty ratio. We generated constitutively active motor domain constructs of two class-1 myosins from the social amoeba , namely, Myo1E, a high duty ratio myosin and Myo1B, a low duty ratio myosin. In Myo1E we introduced mutation Y388F and in Myo1B mutation F387Y. The detailed functional characterization by steady-state and transient kinetic experiments, combined with motility and landing assays revealed an almost reciprocal relationship of a number of critical kinetic parameters and equilibrium constants between wild-type and mutants that dictate the lifetime of the strongly actin-attached states of myosin. The Y-to-F mutation increased the duty ratio of Moy1B by almost one order of magnitude, while the introduction of the phenylalanine in switch-2 of Myo1E transformed the myosin into a low duty ratio motor. These data together with structural considerations propose a role of switch-2 in fine-tuning ADP release through a mechanism, where the class-specific tyrosine together with surrounding residues contributes to the coordination of Mg and ADP. Our results highlight the importance of conserved switch-2 residues in class-1 myosins for efficient chemo-mechanical coupling, revealing that switch-2 is important to adjust the duty ratio of the amoeboid class-1 myosins for performing movement, transport or gating functions.
PubMed: 38887318
DOI: 10.3389/fphys.2024.1393952 -
Journal of Microbiology (Seoul, Korea) Jun 2024Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are...
Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins.
PubMed: 38884692
DOI: 10.1007/s12275-024-00143-y -
Life Sciences Aug 2024Differentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of...
Mammalian target of differentiation-inducing factor-1 is mitochondrial malate dehydrogenase for activation of AMP-activated protein kinase and induction of mitochondrial fission.
AIMS
Differentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells.
MAIN METHODS
To examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells.
KEY FINDINGS
DIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission.
SIGNIFICANCE
We propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.
Topics: Humans; AMP-Activated Protein Kinases; Mitochondrial Dynamics; Malate Dehydrogenase; Mitochondria; HeLa Cells; Animals; Hexanones; Human Umbilical Vein Endothelial Cells; Enzyme Activation; Hydrocarbons, Chlorinated
PubMed: 38852800
DOI: 10.1016/j.lfs.2024.122807 -
Natural Product Research Jun 2024The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to...
The rise of antibiotic-resistant bacterial strains represents an important challenge for global health, underscoring the critical need for innovative strategies to confront this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba is a potent model organism in this effort. Employing this invertebrate model, we introduce a novel perspective to investigate natural plant extracts in search of molecules with potential antivirulence activity. Our work established an easy-scalable developmental assay targeting a virulent strain of , with as the representative plant. The main objective was to identify tentative compounds from the extract that attenuate the virulence of virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of fulfilled these prerequisites compared to the dichloromethane extract. Using UHPLC Q/Orbitrap/ESI/MS/MS, 63 compounds were tentatively identified in both extracts, 47 in the methanolic and 29 in the dichloromethane, with 13 compounds in common. This research underscores the potential of employing -assisted pharmacognosy to discover new antivirulence agents against multidrug-resistant pathogens.
PubMed: 38829280
DOI: 10.1080/14786419.2024.2360166 -
Frontiers in Physiology 2024I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the motility assay and the dual-beam... (Review)
Review
I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the motility assay and the dual-beam single-molecule laser trap assay for myosin-driven actin filament movement, used widely as key assays for understanding how both muscle and nonmuscle myosin molecular motors work. As for all discoveries, the history of the development of the myosin assays involves many people who are not authors of the final publications, but without whom the assays would not have been developed as they are. Also, early experiences shape how one develops ideas and experiments, and influence future discoveries in major ways. I am pleased here to trace my own path and acknowledge the many individuals involved and my early science experiences that led to the work I and my students, postdoctoral fellows, and sabbatical visitors did to develop these assays. Mentors are too often overlooked in historical descriptions of discoveries, and my story starts with those who mentored me.
PubMed: 38827995
DOI: 10.3389/fphys.2024.1390186 -
Evolution Letters Jun 2024In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution...
In facultative symbioses, only a fraction of hosts are associated with symbionts. Specific host and symbiont pairings may be the result of host-symbiont coevolution driven by reciprocal selection or priority effects pertaining to which potential symbiont is associated with a host first. Distinguishing between these possibilities is important for understanding the evolutionary forces that affect facultative symbioses. We used the social amoeba, , and its symbiont, , to determine whether ongoing coevolution affects which host-symbiont strain pairs naturally cooccur within a facultative symbiosis. Relative to other , including another symbiont of , features a reduced genome size that indicates a significant history of coevolution with its host. We hypothesized that ongoing host-symbiont coevolution would lead to higher fitness for naturally cooccurring (native) host and symbiont pairings compared to novel pairings. We show for the first time that symbionts can horizontally transmit to new amoeba hosts when hosts aggregate together during the social stage of their life cycle. Here we find evidence for a virulence-transmission trade-off without host specificity. Although symbiont strains were significantly variable in virulence and horizontal transmission rate, hosts and symbionts responded similarly to associations in native and novel pairings. We go on to identify candidate virulence factors in the genomes of strains that may contribute to variation in virulence. We conclude that ongoing coevolution is unlikely for and The system instead appears to represent a stable facultative symbiosis in which naturally cooccurring host and symbiont pairings are the result of priority effects.
PubMed: 38818420
DOI: 10.1093/evlett/qrae001 -
The Journal of Biological Chemistry May 2024Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested...
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
PubMed: 38815864
DOI: 10.1016/j.jbc.2024.107423 -
PeerJ 2024The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been...
The evolution of symbiotic interactions may be affected by unpredictable conditions. However, a link between prevalence of these conditions and symbiosis has not been widely demonstrated. We test for these associations using social amoebae and their bacterial endosymbionts. commonly hosts endosymbiotic bacteria from three taxa: and Chlamydiae. Three species of facultative endosymbionts are the best studied and give hosts the ability to carry prey bacteria through the dispersal stage to new environments. and Chlamydiae are obligate endosymbiont lineages with no measurable impact on host fitness. We tested whether the frequency of both single infections and coinfections of these symbionts were associated with the unpredictability of their soil environments by using symbiont presence-absence data from isolates from 21 locations across the eastern United States. We found that symbiosis across all infection types, symbiosis with and Chlamydiae obligate endosymbionts, and symbiosis involving coinfections were not associated with any of our measures. However, unpredictable precipitation was associated with symbiosis in two species of , suggesting a link between unpredictable conditions and symbiosis.
Topics: Symbiosis; Soil Microbiology; Dictyostelium; Burkholderiaceae; Soil; United States; Chlamydia
PubMed: 38784393
DOI: 10.7717/peerj.17445