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International Journal of Molecular... Dec 2023In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps....
In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps. Our approach was designed for highly complex intracellular architectures in which a long-range cytoskeleton network extends throughout the cell bodies and protrusions. The irregular organization of the actin network, as well as cryo-electron-tomography-specific noise, missing wedge artifacts, and map dimensions call for a specialized implementation that is both robust and efficient. Our proposed solution, , accumulates densities along paths of a specific length in various directions, starting from locally determined seed points. The highest-density paths originating from the seed points form short linear candidate filament segments, which are further scrutinized and classified by users via inspection of a novel , which visualizes the likelihood of being a part of longer filaments. The pruned linear candidate filament segments are then iteratively fused into continuous, longer, and curved filaments based on their relative orientations, gap spacings, and extendibility. When applied to the simulated phantom tomograms of a filopodium under experimental conditions, demonstrated high efficacy, with F1-scores ranging from 0.85 to 0.90, depending on the noise level. Furthermore, when applied to a previously untraced experimental tomogram of mouse fibroblast lamellipodia, the filaments predicted by exhibited a good visual agreement with the experimental map. The framework is highly time efficient and can complete the tracing process in just a few minutes. The source code is publicly available with version 3.2 of the free and open-source software package.
Topics: Mice; Animals; Dictyostelium; Actin Cytoskeleton; Cytoskeleton; Actins; Electron Microscope Tomography
PubMed: 38139012
DOI: 10.3390/ijms242417183 -
Journal of Cell Science Jul 2023During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of...
During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.
Topics: Dictyostelium; Phosphates; Protozoan Proteins; Cell Proliferation; Genetic Testing
PubMed: 37259831
DOI: 10.1242/jcs.260541 -
BioRxiv : the Preprint Server For... Oct 2023Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene...
Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear if these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.
PubMed: 37398420
DOI: 10.1101/2023.05.31.543154 -
Scientific Reports Sep 2023Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded...
Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory 'switch helix' that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.
Topics: Humans; Animals; Saccharomyces cerevisiae; Calcium; Dictyostelium; Actins; Calcium, Dietary; Schizosaccharomyces; Arabidopsis; Mammals
PubMed: 37758724
DOI: 10.1038/s41598-023-42682-1 -
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 -
ELife Feb 2024Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many...
Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including RNA-mediated transposition and pseudohyphal growth, filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, xylan catabolism and meiosis, development and sexual cycles, infection, and the nervous systems of and . We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAA and TAG codons. Notably, the usage frequency of CAG is much lower in ciliates with reassigned TAA and TAG codons than in organisms with expanded and unstable Q runs (e.g. and ), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.
Topics: Animals; Mice; Codon, Terminator; Drosophila melanogaster; Dictyostelium; Fungal Proteins; Glutamine
PubMed: 38393970
DOI: 10.7554/eLife.91405 -
Development (Cambridge, England) Dec 2023Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it...
Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.
Topics: Dictyostelium; Signal Transduction; Transcriptome; Gene Expression Profiling
PubMed: 37921687
DOI: 10.1242/dev.201946 -
Genes To Cells : Devoted To Molecular &... Dec 2023Cytokinesis, the final process of cell division, involves the accumulation of actin and myosin II filaments at the cell's equator, forming a contractile ring that...
Cytokinesis, the final process of cell division, involves the accumulation of actin and myosin II filaments at the cell's equator, forming a contractile ring that facilitates the division into two daughter cells. While light microscopy has provided valuable insights into the molecular mechanism of this process, it has limitations in examining individual filaments in vivo. In this study, we utilized transmission electron microscopy to observe actin and myosin II filaments in the contractile rings of dividing Dictyostelium cells. To synchronize cytokinesis, we developed a novel method that allowed us to visualize dividing cells undergoing cytokinesis with a frequency as high as 18%. This improvement enabled us to examine the lengths and alignments of individual filaments within the contractile rings. As the furrow constricted, the length of actin filaments gradually decreased. Moreover, both actin and myosin II filaments reoriented perpendicularly to the long axis during furrow constriction. Through experiments involving myosin II null cells, we discovered that myosin II plays a role in regulating both the lengths and alignments of actin filaments. Additionally, dynamin-like protein A was found to contribute to regulating the length of actin filaments, while cortexillins were involved in regulating their alignment.
Topics: Actomyosin; Actins; Dictyostelium; Actin Cytoskeleton; Cytokinesis; Myosin Type II
PubMed: 37844904
DOI: 10.1111/gtc.13073 -
Biophysical Journal Aug 2023Identifying the directionality of signaling sources from noisy input to membrane receptors is an essential task performed by many cell types. A variety of models have...
Identifying the directionality of signaling sources from noisy input to membrane receptors is an essential task performed by many cell types. A variety of models have been proposed to explain directional sensing in cells. However, many of these require significant computational and memory capacities for the cell. We propose and analyze a simple mechanism in which a cell adopts the direction associated with the first few membrane binding events. This model yields an accurate angular estimate to the source long before steady state is reached in biologically relevant scenarios. Our proposed mechanism allows for reliable estimates of the directionality of external signals using temporal information and assumes minimal computational capacities of the cell.
Topics: Signal Transduction; Dictyostelium
PubMed: 37355773
DOI: 10.1016/j.bpj.2023.06.015 -
MBio Oct 2023Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic species secrete...
Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe . This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in . In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as .
Topics: Humans; Polyphosphates; Diphosphates; Dictyostelium; Bacteria; Phagocytosis; TOR Serine-Threonine Kinases
PubMed: 37754562
DOI: 10.1128/mbio.01939-23