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The Journal of Cell Biology Oct 2024Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes...
Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.
Topics: Animals; Actin-Related Protein 2-3 Complex; Actins; Cell Membrane; Centrosome; Drosophila melanogaster; Drosophila Proteins; Dyneins; Exocytosis; Microtubules
PubMed: 38935075
DOI: 10.1083/jcb.202403115 -
Life Science Alliance Sep 2024Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation...
Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of sperm and the abnormal movement of left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.
Topics: Animals; Zebrafish; Male; Calcium; Spermatozoa; Animals, Genetically Modified; Zebrafish Proteins; Dyneins; Cilia; Flagella; Sperm Motility; Calcium-Binding Proteins
PubMed: 38876797
DOI: 10.26508/lsa.202402632 -
The Journal of Cell Biology Sep 2024At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro,...
At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are together required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.
Topics: Humans; Anaphase; Spindle Apparatus; Kinesins; Microtubules; Dyneins; Torque; Chromosome Segregation; Actin Cytoskeleton; Microtubule-Associated Proteins
PubMed: 38869473
DOI: 10.1083/jcb.202312046 -
The Journal of Cell Biology Sep 2024Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule...
Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.
Topics: Axoneme; Biological Transport; Cilia; Dyneins; Flagella; Kinesins; Leishmania; Protozoan Proteins; Microtubules
PubMed: 38829962
DOI: 10.1083/jcb.202401154 -
Molecular Biology of the Cell Jul 2024Tubulins undergo several kinds of posttranslational modifications (PTMs) including glutamylation and glycylation. The contribution of these PTMs to the motilities of...
Tubulins undergo several kinds of posttranslational modifications (PTMs) including glutamylation and glycylation. The contribution of these PTMs to the motilities of cilia and flagella is still unclear. Here, we investigated the role of tubulin glycylation by examining a novel mutant lacking TTLL3, an enzyme responsible for initiating glycylation. Immunostaining of cells and flagella revealed that glycylation is only restricted to the axonemal tubulin composing the outer-doublet but not the central-pair microtubules. Furthermore, the flagellar localization of TTLL3 was found to be dependent on intraflagellar transport. The mutant, , completely lacks glycylation and consequently exhibits slower swimming velocity compared with the wild-type strain. By combining the mutation with multiple axonemal dynein-deficient mutants, we found that the lack of glycylation does not affect the motility of the outer-arm dynein lacking mutations. Sliding disintegration assay using isolated axonemes revealed that the lack of glycylation decreases microtubule sliding velocity in the normal axoneme but not in the axoneme lacking the outerarm dyneins. Based on our recent study that glycylation occurs exclusively on β-tubulin in , these findings suggest that tubulin glycylation controls flagellar motility through modulating outer-arm dyneins, presumably by neutralizing the negative charges of glutamate residues at the C-terminus region of β-tubulin.
Topics: Cilia; Tubulin; Flagella; Axoneme; Protein Processing, Post-Translational; Microtubules; Chlamydomonas reinhardtii; Dyneins; Chlamydomonas; Mutation; Axonemal Dyneins
PubMed: 38758663
DOI: 10.1091/mbc.E24-04-0154 -
Life Science Alliance Jul 2024Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6-positive vesicles for...
Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6 by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6 that abolish binding to BicD2 also result in reduced co-migration of Rab6/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.
Topics: rab GTP-Binding Proteins; Humans; Dyneins; Protein Binding; Binding Sites; Kinesins; Mutation; Microtubule-Associated Proteins; Protein Transport; Models, Molecular; Guanosine Triphosphate
PubMed: 38719748
DOI: 10.26508/lsa.202302430 -
The Journal of Biological Chemistry Apr 2024Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different...
Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
PubMed: 38677516
DOI: 10.1016/j.jbc.2024.107323 -
Nature Communications Apr 2024Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport...
Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.
Topics: Caenorhabditis elegans; Animals; Cilia; Caenorhabditis elegans Proteins; Kinesins; Flagella; Tubulin; Axoneme; Dyneins; Biological Transport; Single Molecule Imaging; Protein Transport
PubMed: 38658528
DOI: 10.1038/s41467-024-47807-2 -
BMC Medical Genomics Apr 2024Liver cancer ranks sixth in incidence and third in mortality globally and hepatocellular carcinoma (HCC) accounts for 90% of it. Hypoxia, glycolysis, and lactate...
BACKGROUND
Liver cancer ranks sixth in incidence and third in mortality globally and hepatocellular carcinoma (HCC) accounts for 90% of it. Hypoxia, glycolysis, and lactate metabolism have been found to regulate the progression of HCC separately. However, there is a lack of studies linking the above three to predict the prognosis of HCC. The present study aimed to identify a hypoxia-glycolysis-lactate-related gene signature for assessing the prognosis of HCC.
METHODS
This study collected 510 hypoxia-glycolysis-lactate genes from Molecular Signatures Database (MSigDB) and then classified HCC patients from TCGA-LIHC by analyzing their hypoxia-glycolysis-lactate genes expression. Differentially expressed genes (DEGs) were screened out to construct a gene signature by LASSO-Cox analysis. Univariate and multivariate regression analyses were used to evaluate the independent prognostic value of the gene signature. Analyses of immune infiltration, somatic cell mutations, and correlation heatmap were conducted by "GSVA" R package. Single-cell analysis conducted by "SingleR", "celldex", "Seurat", and "CellCha" R packages revealed how signature genes participated in hypoxia/glycolysis/lactate metabolism and PPI network identified hub genes.
RESULTS
We classified HCC patients from TCGA-LIHC into two clusters and screened out DEGs. An 18-genes prognostic signature including CDCA8, CBX2, PDE6A, MED8, DYNC1LI1, PSMD1, EIF5B, GNL2, SEPHS1, CCNJL, SOCS2, LDHA, G6PD, YBX1, RTN3, ADAMTS5, CLEC3B, and UCK2 was built to stratify the risk of HCC. The risk score of the hypoxia-glycolysis-lactate gene signature was further identified as a valuable independent factor for estimating the prognosis of HCC. Then we found that the features of clinical characteristics, immune infiltration, somatic cell mutations, and correlation analysis differed between the high-risk and low-risk groups. Furthermore, single-cell analysis indicated that the signature genes could interact with the ligand-receptors of hepatocytes/fibroblasts/plasma cells to participate in hypoxia/glycolysis/lactate metabolism and PPI network identified potential hub genes in this process: CDCA8, LDHA, YBX1.
CONCLUSION
The hypoxia-glycolysis-lactate-related gene signature we built could provide prognostic value for HCC and suggest several hub genes for future HCC studies.
Topics: Humans; Lactic Acid; Carcinoma, Hepatocellular; Liver Neoplasms; Prognosis; Hypoxia; Eye Proteins; Cyclic Nucleotide Phosphodiesterases, Type 6; Cytoplasmic Dyneins
PubMed: 38627714
DOI: 10.1186/s12920-024-01867-x -
Lung Jun 2024We aimed to examine the correlation between clinical characteristics and the pathogenic gene variants in patients with Primary Ciliary Dyskinesia (PCD).
PURPOSE
We aimed to examine the correlation between clinical characteristics and the pathogenic gene variants in patients with Primary Ciliary Dyskinesia (PCD).
METHODS
We conducted a retrospective single-center study in patients with PCD followed at the University Hospitals Leuven. We included patients with genetically confirmed PCD and described their genotype, data from ultrastructural ciliary evaluation and clinical characteristics. Genotype/phenotype correlations were studied in patients with the most frequently involved genes.
RESULTS
We enrolled 74 patients with a median age of 25.58 years. The most frequently involved genes were DNAH11 (n = 23) and DNAH5 (n = 19). The most frequent types of pathogenic variants were missense (n = 42) and frameshift variants (n = 36) and most patients had compound heterozygous variants (n = 44). Ciliary ultrastructure (p < 0.001), situs (p = 0.015) and age at diagnosis (median 9.50 vs 4.71 years, p = 0.037) differed between DNAH11 and DNAH5. When correcting for situs this difference in age at diagnosis was no longer significant (p = 0.973). Patients with situs inversus were diagnosed earlier (p = 0.031). Respiratory tract microbiology (p = 0.161), lung function (cross-sectional, p = 0.829 and longitudinal, p = 0.329) and chest CT abnormalities (p = 0.202) were not significantly different between DNAH11 and DNAH5 variants.
CONCLUSION
This study suggests a genotype-phenotype correlation for some of the evaluated clinical characteristics of the two most frequently involved genes in this study, namely DNAH11 and DNAH5.
Topics: Humans; Male; Female; Adult; Retrospective Studies; Belgium; Child; Adolescent; Child, Preschool; Young Adult; Axonemal Dyneins; Dyneins; Middle Aged; Kartagener Syndrome; Genetic Association Studies; Phenotype; Infant; Situs Inversus; Cilia; Mutation, Missense; Frameshift Mutation
PubMed: 38602513
DOI: 10.1007/s00408-024-00696-0