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Scientific Reports Sep 2022The Cafeteria roenbergensis virus (Crov), Dictyostelium, and other species encode a large family of leucine-rich repeat (LRR) proteins with FGxxFN motifs. We determined...
The Cafeteria roenbergensis virus (Crov), Dictyostelium, and other species encode a large family of leucine-rich repeat (LRR) proteins with FGxxFN motifs. We determined the structures of two of them and observed several unique structural features that set them aside from previously characterized LRR family members. Crov588 comprises 25 regular repeats with a LxxLxFGxxFNQxIxENVLPxx consensus, forming a unique closed circular repeat structure. Novel features include a repositioning of a conserved asparagine at the middle of the repeat, a double phenylalanine spine that generates an alternate core packing arrangement, and a histidine/tyrosine ladder on the concave surface. Crov539 is smaller, comprising 12 repeats of a similar LxxLxFGxxFNQPIExVxW/LPxx consensus and forming an unusual cap-swapped dimer structure. The phenylalanine spine of Crov539 is supplemented with a tryptophan spine, while a hydrophobic isoleucine-rich patch is found on the central concave surface. We present a detailed analysis of the structures of Crov588 and Crov539 and compare them to related repeat proteins and other LRR classes.
Topics: Amino Acid Sequence; Asparagine; Dictyostelium; Histidine; Isoleucine; Leucine; Leucine-Rich Repeat Proteins; Phenylalanine; Tryptophan; Tyrosine
PubMed: 36180492
DOI: 10.1038/s41598-022-20758-8 -
Frontiers in Cell and Developmental... 2021WIPIs are a conserved family of proteins with a characteristic 7-bladed β-propeller structure. They play a prominent role in autophagy, but also in other membrane... (Review)
Review
WIPIs are a conserved family of proteins with a characteristic 7-bladed β-propeller structure. They play a prominent role in autophagy, but also in other membrane trafficking processes. Mutations in human cause several neurodegenerative diseases. One of them is BPAN, a rare disease characterized by developmental delay, motor disorders, and seizures. Autophagy dysfunction is thought to play an important role in this disease but the precise pathological consequences of the mutations are not well established. The use of simple models such as the yeast and the social amoeba provides valuable information on the molecular and cellular function of these proteins, but also sheds light on possible pathways that may be relevant in the search for potential therapies. Here, we review the function of WIPIs as well as disease-causing mutations with a special focus on the information provided by these simple models.
PubMed: 34540850
DOI: 10.3389/fcell.2021.737071 -
Proceedings of the National Academy of... Dec 2021In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite...
In fast-moving cells such as amoeba and immune cells, dendritic actin filaments are spatiotemporally regulated to shape large-scale plasma membrane protrusions. Despite their importance in migration, as well as in particle and liquid ingestion, how their dynamics are affected by micrometer-scale features of the contact surface is still poorly understood. Here, through quantitative image analysis of on microfabricated surfaces, we show that there is a distinct mode of topographical guidance directed by the macropinocytic membrane cup. Unlike other topographical guidance known to date that depends on nanometer-scale curvature sensing protein or stress fibers, the macropinocytic membrane cup is driven by the Ras/PI3K/F-actin signaling patch and its dependency on the micrometer-scale topographical features, namely PI3K/F-actin-independent accumulation of Ras-GTP at the convex curved surface, PI3K-dependent patch propagation along the convex edge, and its actomyosin-dependent constriction at the concave edge. Mathematical model simulations demonstrate that the topographically dependent initiation, in combination with the mutually defining patch patterning and the membrane deformation, gives rise to the topographical guidance. Our results suggest that the macropinocytic cup is a self-enclosing structure that can support liquid ingestion by default; however, in the presence of structured surfaces, it is directed to faithfully trace bent and bifurcating ridges for particle ingestion and cell guidance.
Topics: Cell Membrane; Chemotaxis; Computer Simulation; Dictyostelium; Models, Biological; Movement; Phosphatidylinositol 3-Kinases; Pinocytosis; Signal Transduction
PubMed: 34876521
DOI: 10.1073/pnas.2110281118 -
Trends in Microbiology May 2024Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability... (Review)
Review
Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.
Topics: Metals; Phagocytosis; Dictyostelium; Biological Evolution; Acanthamoeba; Animals; Phagosomes; Zinc; Metalloids; Copper; Biological Availability; Mitochondria
PubMed: 38103995
DOI: 10.1016/j.tim.2023.11.008 -
Journal of Cell Science Oct 2023Mitogen-activated protein kinases (MAPKs) have been the focus of many studies over the past several decades, but the understanding of one subgroup of MAPKs, orthologs of... (Review)
Review
Mitogen-activated protein kinases (MAPKs) have been the focus of many studies over the past several decades, but the understanding of one subgroup of MAPKs, orthologs of MAPK15, known as atypical MAPKs, has lagged behind others. In most organisms, specific activating signals or downstream responses of atypical MAPK signaling pathways have not yet been identified even though these MAPKs are associated with many eukaryotic processes, including cancer and embryonic development. In this Review, we discuss recent studies that are shedding new light on both the regulation and function of atypical MAPKs in different organisms. In particular, the analysis of the atypical MAPK in the amoeba Dictyostelium discoideum has revealed important roles in chemotactic responses and gene regulation. The rapid and transient phosphorylation of the atypical MAPK in these responses suggest a highly regulated activation mechanism in vivo despite the ability of atypical MAPKs to autophosphorylate in vitro. Atypical MAPK function can also impact the activation of other MAPKs in amoeba. These advances are providing new perspectives on possible MAPK roles in animals that have not been previously considered, and this might lead to the identification of potential targets for regulating cell movement in the treatment of diseases.
Topics: Animals; Amoeba; Dictyostelium; Phosphorylation; MAP Kinase Signaling System; Gene Expression Regulation; Mitogen-Activated Protein Kinase Kinases
PubMed: 37850857
DOI: 10.1242/jcs.261447 -
Developmental Cell Jul 2023Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the...
Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.
Topics: Animals; Mice; Humans; Proto-Oncogene Proteins c-akt; Dictyostelium; Phosphatidylinositol 3-Kinases; Cell Movement; Mechanistic Target of Rapamycin Complex 2; Intercellular Signaling Peptides and Proteins
PubMed: 37220748
DOI: 10.1016/j.devcel.2023.04.019 -
Molecular and Cellular Biochemistry Jul 2019Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in...
Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.
Topics: Caffeine; Chemotaxis; Cyclic AMP; Dictyostelium; Mechanistic Target of Rapamycin Complex 2; Phosphatidylinositol 3-Kinases; Protozoan Proteins; Second Messenger Systems
PubMed: 30879206
DOI: 10.1007/s11010-019-03520-z -
Interactome and evolutionary conservation of Dictyostelid small GTPases and their direct regulators.Small GTPases Jan 2022GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is...
GTP binding proteins known as small GTPases make up one of the largest groups of regulatory proteins and control almost all functions of living cells. Their activity is under, respectively, positive and negative regulation by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which together with their upstream regulators and the downstream targets of the small GTPases form formidable signalling networks. While genomics has revealed the large size of the GTPase, GEF and GAP repertoires, only a small fraction of their interactions and functions have yet been experimentally explored. Dictyostelid social amoebas have been particularly useful in unravelling the roles of many proteins in the Rac-Rho and Ras-Rap families of GTPases in directional cell migration and regulation of the actin cytoskeleton. Genomes and cell-type specific and developmental transcriptomes are available for species that span the 0.5 billion years of evolution of the group from their unicellular ancestors. In this work, we identified all GTPases, GEFs and GAPs from genomes representative of the four major taxon groups and investigated their phylogenetic relationships and evolutionary conservation and changes in their functional domain architecture and in their developmental and cell-type specific expression. We performed a hierarchical cluster analysis of the expression profiles of the ~2000 analysed genes to identify putative interacting sets of GTPases, GEFs and GAPs, which highlight sets known to interact experimentally and many novel combinations. This work represents a valuable resource for research into all fields of cellular regulation.
Topics: Dictyostelium; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Monomeric GTP-Binding Proteins; Phylogeny
PubMed: 34565293
DOI: 10.1080/21541248.2021.1984829 -
Cells Apr 2020Wound repair of cell membrane is a vital physiological phenomenon. We examined wound repair in cells by using a laserporation, which we recently invented. We examined... (Review)
Review
Wound repair of cell membrane is a vital physiological phenomenon. We examined wound repair in cells by using a laserporation, which we recently invented. We examined the influx of fluorescent dyes from the external medium and monitored the cytosolic Ca after wounding. The influx of Ca through the wound pore was essential for wound repair. Annexin and ESCRT components accumulated at the wound site upon wounding as previously described in animal cells, but these were not essential for wound repair in cells. We discovered that calmodulin accumulated at the wound site upon wounding, which was essential for wound repair. The membrane accumulated at the wound site to plug the wound pore by two-steps, depending on Ca influx and calmodulin. From several lines of evidence, the membrane plug was derived from de novo generated vesicles at the wound site. Actin filaments also accumulated at the wound site, depending on Ca influx and calmodulin. Actin accumulation was essential for wound repair, but microtubules were not essential. A molecular mechanism of wound repair will be discussed.
Topics: Animals; Calcium; Calmodulin; Cell Membrane; Dictyostelium; Endosomal Sorting Complexes Required for Transport; Humans; Wound Healing
PubMed: 32340342
DOI: 10.3390/cells9041058 -
Journal of Cell Science Jun 2022Capping protein Arp2/3 myosin I linker (CARMIL) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of capping protein...
Capping protein Arp2/3 myosin I linker (CARMIL) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of capping protein (CP). Here, we characterize CARMIL-GAP (GAP for GTPase-activating protein), a Dictyostelium CARMIL isoform that contains a ∼130 residue insert that, by homology, confers GTPase-activating properties for Rho-related GTPases. Consistent with this idea, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP-null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP-null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that, although both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the nucleotide state of Rho GTPases is often required to drive Rho-dependent biological processes.
Topics: Actin Capping Proteins; Actin Cytoskeleton; Actins; Carrier Proteins; Dictyostelium; GTPase-Activating Proteins; Microfilament Proteins
PubMed: 35583107
DOI: 10.1242/jcs.258704