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European Journal of Cell Biology Dec 2023Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In...
Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism's life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8 conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8 cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.
Topics: Humans; Dictyostelium; Secretome; Membrane Proteins; Mutation; Lysosomes; Membrane Transport Proteins
PubMed: 37742391
DOI: 10.1016/j.ejcb.2023.151361 -
Molecular Biology of the Cell Dec 2023Although the oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently,...
CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells.
Although the oncogene has been extensively studied, new aspects concerning its role and regulation in normal biology and cancer continue to be discovered. Recently, others and we have shown that the mechanistic Target of Rapamycin Complex 2 (mTORC2) is a Ras effector in and mammalian cells. mTORC2 plays evolutionarily conserved roles in cell survival and migration and has been linked to tumorigenesis. Because is often mutated in lung cancer, we investigated whether a Ras-mTORC2 pathway contributes to enhancing the migration of lung cancer cells expressing oncogenic Ras. We used A549 cells and CRISPR/Cas9 to revert the cells' G12S mutation to wild-type and establish A549 revertant (REV) cell lines, which we then used to evaluate the Ras-mediated regulation of mTORC2 and cell migration. Interestingly, our results suggest that K-Ras and mTORC2 promote A549 cell migration but as part of different pathways and independently of Ras's mutational status. Moreover, further characterization of the A549 cells revealed that loss of mutant K-Ras expression for the wild-type protein leads to an increase in cell growth and proliferation, suggesting that the A549 cells have low mutant dependency and that recovering expression of wild-type K-Ras protein increases these cells tumorigenic potential.
Topics: Animals; Humans; Lung Neoplasms; Genes, ras; A549 Cells; Proto-Oncogene Proteins p21(ras); Mechanistic Target of Rapamycin Complex 2; Dictyostelium; Cell Proliferation; Mutation; Cell Line, Tumor; Mammals
PubMed: 37729017
DOI: 10.1091/mbc.E23-05-0152 -
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 -
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 -
Royal Society Open Science Aug 2023Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a...
Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoeba contain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins from aggregates and may play a central role in defence against pathogens. We measured the number and functionality of sentinel cells in aggregates of infected by bacterial endosymbionts in the genus . Infected produced fewer and less functional sentinel cells, suggesting that may interfere with its host's immune system. Despite impaired sentinel cells, however, infected were less sensitive to ethidium bromide toxicity, suggesting that may also have a protective effect on its host. By contrast, infected by did not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship between and , which has considerable potential as a model for the study of symbiosis.
PubMed: 37593719
DOI: 10.1098/rsos.230727 -
Frontiers in Chemistry 2023The dye-decolorizing peroxidase (DyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known...
The dye-decolorizing peroxidase (DyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DyP catalyzes the HO-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DyP active site and its implication on DyP catalysis.
PubMed: 37593106
DOI: 10.3389/fchem.2023.1220543 -
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 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061 -
ELife Jul 2023Actin dynamics in cell motility, division, and phagocytosis is regulated by complex factors with multiple feedback loops, often leading to emergent dynamic patterns in...
Actin dynamics in cell motility, division, and phagocytosis is regulated by complex factors with multiple feedback loops, often leading to emergent dynamic patterns in the form of propagating waves of actin polymerization activity that are poorly understood. Many in the actin wave community have attempted to discern the underlying mechanisms using experiments and/or mathematical models and theory. Here, we survey methods and hypotheses for actin waves based on signaling networks, mechano-chemical effects, and transport characteristics, with examples drawn from , human neutrophils, , and oocytes. While experimentalists focus on the details of molecular components, theorists pose a central question of universality: Are there generic, model-independent, underlying principles, or just boundless cell-specific details? We argue that mathematical methods are equally important for understanding the emergence, evolution, and persistence of actin waves and conclude with a few challenges for future studies.
Topics: Humans; Actins; Dictyostelium; Cell Movement; Signal Transduction; Phagocytosis; Models, Biological; Actin Cytoskeleton
PubMed: 37428017
DOI: 10.7554/eLife.87181 -
Cells May 2023The Amoebozoan exhibits a semi-closed mitosis in which the nuclear membranes remain intact but become permeabilized to allow tubulin and spindle assembly factors to...
The Amoebozoan exhibits a semi-closed mitosis in which the nuclear membranes remain intact but become permeabilized to allow tubulin and spindle assembly factors to access the nuclear interior. Previous work indicated that this is accomplished at least by partial disassembly of nuclear pore complexes (NPCs). Further contributions by the insertion process of the duplicating, formerly cytosolic, centrosome into the nuclear envelope and nuclear envelope fenestrations forming around the central spindle during karyokinesis were discussed. We studied the behavior of several nuclear envelope, centrosomal, and nuclear pore complex (NPC) components tagged with fluorescence markers together with a nuclear permeabilization marker (NLS-TdTomato) by live-cell imaging. We could show that permeabilization of the nuclear envelope during mitosis occurs in synchrony with centrosome insertion into the nuclear envelope and partial disassembly of nuclear pore complexes. Furthermore, centrosome duplication takes place after its insertion into the nuclear envelope and after initiation of permeabilization. Restoration of nuclear envelope integrity usually occurs long after re-assembly of NPCs and cytokinesis has taken place and is accompanied by a concentration of endosomal sorting complex required for transport (ESCRT) components at both sites of nuclear envelope fenestration (centrosome and central spindle).
Topics: Nuclear Envelope; Dictyostelium; Amoeba; Mitosis; Cell Nucleus
PubMed: 37408214
DOI: 10.3390/cells12101380