-
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 -
Frontiers in Cell and Developmental... 2023Spatial and temporal variations of oxygen environments affect the behaviors of various cells and are involved in physiological and pathological events. Our previous...
Spatial and temporal variations of oxygen environments affect the behaviors of various cells and are involved in physiological and pathological events. Our previous studies with as a model of cell motility have demonstrated that aerotaxis toward an oxygen-rich region occurs below 2% O. However, while the aerotaxis of seems to be an effective strategy to search for what is essential for survival, the mechanism underlying this phenomenon is still largely unclear. One hypothesis is that an oxygen concentration gradient generates a secondary oxidative stress gradient that would direct cell migration towards higher oxygen concentration. Such mechanism was inferred but not fully demonstrated to explain the aerotaxis of human tumor cells. Here, we investigated the role on aerotaxis of flavohemoglobins, proteins that can both act as potential oxygen sensors and modulators of nitric oxide and oxidative stress. The migratory behaviors of cells were observed under both self-generated and imposed oxygen gradients. Furthermore, their changes by chemicals generating or preventing oxidative stress were tested. The trajectories of the cells were then analyzed through time-lapse phase-contrast microscopic images. The results indicate that both oxidative and nitrosative stresses are not involved in the aerotaxis of but cause cytotoxic effects that are enhanced upon hypoxia.
PubMed: 37397260
DOI: 10.3389/fcell.2023.1134011 -
Frontiers in Cellular and Infection... 2023
Topics: Animals; Communicable Diseases; Disease Models, Animal
PubMed: 37389208
DOI: 10.3389/fcimb.2023.1225129 -
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 -
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 -
Microorganisms May 2023Recent studies have shown that can survive in different environments, including soils, and they can maintain populations in sterile soil for a long period of time. This...
Recent studies have shown that can survive in different environments, including soils, and they can maintain populations in sterile soil for a long period of time. This indicates that growth-supporting nutrients are available; however, when grown in non-sterile soils, populations decline, suggesting that other biological factors play a role in controlling populations in soil. Free-living protozoa can affect the bacterial population by grazing. We hypothesized that strains capable of surviving in non-sterile soil possess mechanisms to protect themselves from amoeba predation. We determined the grazing rate of pasture isolates by using . Bacterial suspensions applied to lactose agar as lines were allowed to grow for 24 h, when 4 μL of culture was inoculated in the center of each bacterial line. Grazing distances were measured after 4 days. The genomes of five grazing-susceptible and five grazing-resistant isolates were sequenced and compared. Grazing distance varied among isolates, which indicated that some are more susceptible to grazing by protozoa than others. When presented with a choice between grazing-susceptible and grazing-resistant isolates, grazed only on the susceptible strain. Grazing susceptibility phenotype did not align with the phylogroup, with both B1 and E strains found in both grazing groups. They also did not align by core genome phylogeny. Whole genome comparisons revealed that the five most highly grazed strains had 389 shared genes not found in the five least grazed strains. Conversely, the five least grazed strains shared 130 unique genes. The results indicate that long-term persistence of in soil is due at least in part to resistance to grazing by soil amoeba.
PubMed: 37374960
DOI: 10.3390/microorganisms11061457 -
Biology Jun 2023Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate...
Differentiation-inducing factors 1 and 2 (DIF-1 and DIF-2) are small lipophilic signal molecules that induce stalk cell differentiation but differentially modulate chemotaxis toward cAMP in the cellular slime mold ; DIF-1 suppresses chemotactic cell movement in shallow cAMP gradients, whereas DIF-2 promotes it. The receptor(s) for DIF-1 and DIF-2 have not yet been identified. We examined the effects of nine derivatives of DIF-1 on chemotactic cell movement toward cAMP and compared their chemotaxis-modulating activity and stalk cell differentiation-inducing activity in wild-type and mutant strains. The DIF derivatives differentially affected chemotaxis and stalk cell differentiation; for example, TM-DIF-1 suppressed chemotaxis and showed poor stalk-inducing activity, DIF-1(3M) suppressed chemotaxis and showed strong stalk-inducing activity, and TH-DIF-1 promoted chemotaxis. These results suggest that DIF-1 and DIF-2 have at least three receptors: one for stalk cell induction and two for chemotaxis modulation. In addition, our results show that the DIF derivatives can be used to analyze the DIF-signaling pathways in .
PubMed: 37372157
DOI: 10.3390/biology12060873 -
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 -
Cells May 2023Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is...
Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba . In comparison to AX2 wild-type cells, ATG9and ATG16 cells displayed a 60%, and ATG9/16 cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9, ATG16, and ATG9/16 cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16 cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis.
Topics: Proteasome Endopeptidase Complex; Dictyostelium; Proteomics; Autophagy; Transcription Factors; Ubiquitin
PubMed: 37296637
DOI: 10.3390/cells12111514 -
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