-
Frontiers in Genetics 2022Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin... (Review)
Review
Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes' CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in and , fourteen in , one in and . CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.
PubMed: 35186026
DOI: 10.3389/fgene.2022.781885 -
ACS Nano May 2023Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope....
Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.
Topics: Humans; Microscopy, Fluorescence; Dictyostelium; Energy Transfer; Fluorescent Dyes
PubMed: 36995274
DOI: 10.1021/acsnano.2c12372 -
Biophysical Journal Jul 2022Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and...
Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.
Topics: Actins; Cell Movement; Chemotactic Factors; Chemotaxis; Dictyostelium; Myosin Type II; Pseudopodia
PubMed: 35644945
DOI: 10.1016/j.bpj.2022.05.043 -
Biological Reviews of the Cambridge... Apr 2020This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of... (Review)
Review
This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin-binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin-dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin-dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin-dependent transdifferentiation to re-establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin-dependent germination of spores. Specific calmodulin-binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.
Topics: Calcium Signaling; Calmodulin; Cell Movement; Chemotaxis; Dictyostelium; Life Cycle Stages
PubMed: 31774219
DOI: 10.1111/brv.12573 -
Nucleic Acids Research Jan 2021The Gene Ontology Consortium (GOC) provides the most comprehensive resource currently available for computable knowledge regarding the functions of genes and gene...
The Gene Ontology Consortium (GOC) provides the most comprehensive resource currently available for computable knowledge regarding the functions of genes and gene products. Here, we report the advances of the consortium over the past two years. The new GO-CAM annotation framework was notably improved, and we formalized the model with a computational schema to check and validate the rapidly increasing repository of 2838 GO-CAMs. In addition, we describe the impacts of several collaborations to refine GO and report a 10% increase in the number of GO annotations, a 25% increase in annotated gene products, and over 9,400 new scientific articles annotated. As the project matures, we continue our efforts to review older annotations in light of newer findings, and, to maintain consistency with other ontologies. As a result, 20 000 annotations derived from experimental data were reviewed, corresponding to 2.5% of experimental GO annotations. The website (http://geneontology.org) was redesigned for quick access to documentation, downloads and tools. To maintain an accurate resource and support traceability and reproducibility, we have made available a historical archive covering the past 15 years of GO data with a consistent format and file structure for both the ontology and annotations.
Topics: Animals; Arabidopsis; Caenorhabditis elegans; Dictyostelium; Drosophila melanogaster; Escherichia coli; Gene Ontology; Humans; Internet; Mice; Molecular Sequence Annotation; Rats; Saccharomyces cerevisiae; Schizosaccharomyces; User-Computer Interface; Zebrafish
PubMed: 33290552
DOI: 10.1093/nar/gkaa1113 -
Frontiers in Genetics 2022Mutations in cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of...
Mutations in cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. The NCLs, commonly referred to as Batten disease, are a family of neurodegenerative lysosomal storage diseases that affect all ages and ethnicities globally. Previous research showed that CLN5 participates in a variety of cellular processes. However, the precise function of CLN5 in the cell and the pathway(s) regulating its function are not well understood. In the model organism , loss of the homolog, , impacts various cellular and developmental processes including cell proliferation, cytokinesis, aggregation, cell adhesion, and terminal differentiation. In this study, we used comparative transcriptomics to identify differentially expressed genes underlying -deficiency phenotypes during growth and the early stages of multicellular development. During growth, genes associated with protein ubiquitination/deubiquitination, cell cycle progression, and proteasomal degradation were affected, while genes linked to protein and carbohydrate catabolism were affected during early development. We followed up this analysis by showing that loss of alters the intracellular and extracellular amounts of proliferation repressors during growth and increases the extracellular amount of conditioned medium factor, which regulates cAMP signalling during the early stages of development. Additionally, cells displayed increased intracellular and extracellular amounts of discoidin, which is involved in cell-substrate adhesion and migration. Previous work in mammalian models reported altered lysosomal enzyme activity due to mutation or loss of . Here, we detected altered intracellular activities of various carbohydrate enzymes and cathepsins during growth and starvation. Notably, cells displayed reduced β-hexosaminidase activity, which aligns with previous work showing that Cln5 and human CLN5 can cleave the substrate acted upon by β-hexosaminidase. Finally, consistent with the differential expression of genes associated with proteasomal degradation in cells, we also observed elevated amounts of a proteasome subunit and reduced proteasome 20S activity during growth and starvation. Overall, this study reveals the impact of -deficiency on gene expression in , provides insight on the genes and proteins that play a role in regulating Cln5-dependent processes, and sheds light on the molecular mechanisms underlying CLN5 disease.
PubMed: 36437924
DOI: 10.3389/fgene.2022.1045738 -
BioTechniques Aug 2022We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This...
We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This method yields a substantial amount at microgram levels of gDNA per gel cleanup with high purity. An RNase A treatment step can be omitted. The quality of gDNA is suitable for next-generation sequencing, resulting in >10 Mb reads and high-quality read data (Phred score >28 up to 100 of 150 base reads). Furthermore, the gDNA can be kept intact in a gel slice for several days. This method has been tested for dictyostelids, bacteria and plants.
Topics: Bacteria; DNA; Electrophoresis, Agar Gel; High-Throughput Nucleotide Sequencing
PubMed: 35950336
DOI: 10.2144/btn-2022-0013 -
Frontiers in Cell and Developmental... 2021How phagocytes find invading microorganisms and eliminate pathogenic ones from human bodies is a fundamental question in the study of infectious diseases. About 2.5... (Review)
Review
How phagocytes find invading microorganisms and eliminate pathogenic ones from human bodies is a fundamental question in the study of infectious diseases. About 2.5 billion years ago, eukaryotic unicellular organisms-protozoans-appeared and started to interact with various bacteria. Less than 1 billion years ago, multicellular animals-metazoans-appeared and acquired the ability to distinguish self from non-self and to remove harmful organisms from their bodies. Since then, animals have developed innate immunity in which specialized white-blood cells phagocytes- patrol the body to kill pathogenic bacteria. The social amoebae are prototypical phagocytes that chase various bacteria via chemotaxis and consume them as food via phagocytosis. Studies of this genetically amendable organism have revealed evolutionarily conserved mechanisms underlying chemotaxis and phagocytosis and shed light on studies of phagocytes in mammals. In this review, we briefly summarize important studies that contribute to our current understanding of how phagocytes effectively find and kill pathogens via chemotaxis and phagocytosis.
PubMed: 34490271
DOI: 10.3389/fcell.2021.724940 -
Cells Feb 2020Individual gene analyses of microtubule-based motor proteins in have provided a rough draft of its machinery for cytoplasmic organization and division. This review... (Review)
Review
Individual gene analyses of microtubule-based motor proteins in have provided a rough draft of its machinery for cytoplasmic organization and division. This review collates their activities and looks forward to what is next. A comprehensive approach that considers the collective actions of motors, how they balance rates and directions, and how they integrate with the actin cytoskeleton will be necessary for a complete understanding of cellular dynamics.
Topics: Cell Movement; Dictyostelium; Microtubules; Protozoan Infections; Time Factors
PubMed: 32106406
DOI: 10.3390/cells9030528 -
ELife Aug 2021Using a self-generated hypoxic assay, we show that the amoeba displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume...
Using a self-generated hypoxic assay, we show that the amoeba displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume the available oxygen (O) and form a dense ring moving outwards at constant speed and density. To decipher this collective process, we combined two technological developments: porphyrin-based O -sensing films and microfluidic O gradient generators. We showed that cells exhibit aerotactic and aerokinetic response in a low range of O concentration indicative of a very efficient detection mechanism. Cell behaviors under self-generated or imposed O gradients were modeled using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis.
Topics: Anaerobiosis; Chemotaxis; Dictyostelium; Oxygen
PubMed: 34415238
DOI: 10.7554/eLife.64731