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The Journal of Cell Biology Sep 2023Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key...
Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.
Topics: Animals; Dictyostelium; Endosomes; Mammals; Phagosomes; Phosphatidylinositols; Phosphatidylinositol 3-Kinases
PubMed: 37382666
DOI: 10.1083/jcb.202209077 -
MBio Oct 2023Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate...
Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the / system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as .
Topics: Humans; Vacuoles; Dictyostelium; Endoplasmic Reticulum; Mycobacterium marinum; Mycobacterium tuberculosis; Tuberculosis
PubMed: 37676004
DOI: 10.1128/mbio.00943-23 -
Current Biology : CB Aug 2023Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an...
Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.
Topics: Actins; Dictyostelium; Cell Membrane Structures; Actin Cytoskeleton; Endocytosis
PubMed: 37379843
DOI: 10.1016/j.cub.2023.06.017 -
Frontiers in Cell and Developmental... 2023The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold among eukaryotes. cells...
The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) is only synthesized and utilized by the cellular slime mold among eukaryotes. cells undergo a transition from a unicellular to a multicellular state, ultimately forming a stalk and spores. While is known to employ c-di-GMP to induce differentiation into stalk cells, there have been no reports of direct observation of c-di-GMP using fluorescent probes. In this study, we used a fluorescent probe used in bacteria to visualize its localization within multicellular bodies. Cytosolic c-di-GMP concentrations were significantly higher at the tip of the multicellular body during stalk formation.
PubMed: 37547475
DOI: 10.3389/fcell.2023.1237778 -
The EMBO Journal Dec 2023Motile cells encounter microenvironments with locally heterogeneous mechanochemical composition. Individual compositional parameters, such as chemokines and...
Motile cells encounter microenvironments with locally heterogeneous mechanochemical composition. Individual compositional parameters, such as chemokines and extracellular matrix pore sizes, are well known to provide guidance cues for pathfinding. However, motile cells face diverse cues at the same time, raising the question of how they respond to multiple and potentially competing signals on their paths. Here, we reveal that amoeboid cells require nuclear repositioning, termed nucleokinesis, for adaptive pathfinding in heterogeneous mechanochemical micro-environments. Using mammalian immune cells and the amoeba Dictyostelium discoideum, we discover that frequent, rapid and long-distance nucleokinesis is a basic component of amoeboid pathfinding, enabling cells to reorientate quickly between locally competing cues. Amoeboid nucleokinesis comprises a two-step polarity switch and is driven by myosin-II forces that readjust the nuclear to the cellular path. Impaired nucleokinesis distorts path adaptions and causes cellular arrest in the microenvironment. Our findings establish that nucleokinesis is required for amoeboid cell navigation. Given that many immune cells, amoebae, and some cancer cells utilize an amoeboid migration strategy, these results suggest that nucleokinesis underlies cellular navigation during unicellular biology, immunity, and disease.
Topics: Animals; Cell Movement; Dictyostelium; Amoeba; Extracellular Matrix; Mammals
PubMed: 37987147
DOI: 10.15252/embj.2023114557 -
Cellular Signalling Aug 2023Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying...
Protein kinases are major regulators of cellular processes, but the roles of most kinases remain unresolved. Dictyostelid social amoebas have been useful in identifying functions for 30% of its kinases in cell migration, cytokinesis, vesicle trafficking, gene regulation and other processes but their upstream regulators and downstream effectors are mostly unknown. Comparative genomics can assist to distinguish between genes involved in deeply conserved core processes and those involved in species-specific innovations, while co-expression of genes as evident from comparative transcriptomics can provide cues to the protein complement of regulatory networks. Genomes and developmental and cell-type specific transcriptomes are available for species that span the 0.5 billion years of evolution of Dictyostelia from their unicellular ancestors. In this work we analysed conservation and change in the abundance, functional domain architecture and developmental regulation of protein kinases across the 4 major taxon groups of Dictyostelia. All data are summarized in annotated phylogenetic trees of the kinase subtypes and accompanied by functional information of all kinases that were experimentally studied. We detected 393 different protein kinase domains across the five studied genomes, of which 212 were fully conserved. Conservation was highest (71%) in the previously defined AGC, CAMK, CK1, CMCG, STE and TKL groups and lowest (26%) in the "other" group of typical protein kinases. This was mostly due to species-specific single gene amplification of "other" kinases. Apart from the AFK and α-kinases, the atypical protein kinases, such as the PIKK and histidine kinases were also almost fully conserved. The phylogeny-wide developmental and cell-type specific expression profiles of the protein kinase genes were combined with profiles from the same transcriptomic experiments for the families of G-protein coupled receptors, small GTPases and their GEFs and GAPs, the transcription factors and for all genes that upon lesion generate a developmental defect. This dataset was subjected to hierarchical clustering to identify clusters of co-expressed genes that potentially act together in a signalling network. The work provides a valuable resource that allows researchers to identify protein kinases and other regulatory proteins that are likely to act as intermediates in a network of interest.
Topics: Dictyostelium; Phylogeny; Protein Kinases; Genome; Transcription Factors
PubMed: 37187217
DOI: 10.1016/j.cellsig.2023.110714 -
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 -
Frontiers in Cell and Developmental... 2023Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems and neutrophils over the...
Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems and neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellate . While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement of by employing long-time phase contrast imaging that automatically tracks individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known in and neutrophils. Similarities were also found in the shape dynamics which are characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique to including complex protrusions and the transient bipolar dumbbell morphologies.
PubMed: 38020930
DOI: 10.3389/fcell.2023.1274127 -
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 -
BioRxiv : the Preprint Server For... Sep 2023Studies in the model systems, amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation...
Studies in the model systems, 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.
PubMed: 37693515
DOI: 10.1101/2023.08.30.555648