Model for unidirectional movement of axonemal and cytoplasmic dynein molecules

A model for the unidirectional movement of dynein is presented based on the structuralobservations and biochemical experimental results available.In this model,the binding affinity of dynein formicrotubule (MT) is independent of its nucleotide state and the change between strong and weak MT-bindingis determined naturally by the variation of relative orientation between the stalk and MT,as the stalk rotatesfollowing nucleotide-state transition.Thus the enigmatic communication from the adenosine triphosphate(ATP)-binding site in the globular domain to the far MT-binding site in the tip of the stalk,which is aprerequisite in conventional models,is not required.Using the present model,the previous experimentalresults such as the effect of ATP and adenosine diphosphate (ADP) bindings on dissociation of dynein fromMT,the movement of single-headed axonemal dyneins at saturating ATP concentration,the load dependenceof step-size for the movement of two-headed cytoplasmic dyneins and the dependence of stall force on ATPconcentration can be well explained.     

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.     

The gene for the intermediate chain subunit of cytoplasmic dynein is essential in Drosophila

The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued(1), a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.     

Regulatory dissociation of Tctex-1 light chain from dynein complex is essential for the apical delivery of rhodopsin

Post-Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein-cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex-1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex-1, which mimics Tctex-1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant-negative effect of S82E causes rhodopsin mislocalization in polarized Madin-Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex-1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex-1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.     

Cloning of dynein intermediate chain cDNA of the newt Cynops pyrrhogaster.

To isolate genes whose expression is up-regulated after initiation of meiosis, we employed an mRNA differential display method using RNA extracted from newt testis fragments in the spermatogonial and spermatocyte stages. We report here isolation of a spermatocyte stage-specific cDNA clone encoding a newt homologue of dynein intermediate chain (IC). The newt dynein IC cDNA was found to encode a polypeptide consisting of 694 amino acid residues with 66.8% and 45.8% amino acid sequence similarity to sea urchin dynein IC3 and Chlamydomonas IC69, respectively. The predicted protein contains five WD repeats and a novel repeated motif in the C-terminal region. Northern blot analysis revealed that newt dynein IC mRNA was expressed in the spermatocyte and round spermatid stages, suggesting that dynein IC plays a role in formation of flagella as well as in meiotic events.     

Cloning and characterization of two cytoplasmic dynein intermediate chain genes in mouse and human

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis. Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.     

Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.     

Effect of fuel concentration and force on collective transport by a team of dynein motors

Motor proteins are essential components of intracellular transport inside eukaryotic cells. These protein molecules use chemical energy obtained from hydrolysis of ATP to produce mechanical forces required for transporting cargos inside cells, from one location to another, in a directed manner. Of these motors, cytoplasmic dynein is structurally more complex than other motor proteins involved in intracellular transport, as it shows force and fuel (ATP) concentration dependent step‐size. Cytoplasmic dynein motors are known to work in a team during cargo transport and force generation. Here, we use a complete Monte‐Carlo model of single dynein constrained by in vitro experiments, which includes the effect of both force and ATP on stepping as well as detachment of motors under force. We then use our complete Monte‐Carlo model of single dynein motor to understand collective cargo transport by a team of dynein motors, such as dependence of cargo travel distance and velocity on applied force and fuel concentration. In our model, cargos pulled by a team of dynein motors do not detach rapidly under higher forces, confirming the experimental observation of longer persistence time of dynein team on microtubule under higher forces.     

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.     

Integrating cellular senescence with the concept of damage accumulation in aging: Relevance for clearance of senescent cells

Understanding the aging process and ways to manipulate it is of major importance for biology and medicine. Among the many aging theories advanced over the years, the concept most consistent with experimental evidence posits the buildup of numerous forms of molecular damage as a foundation of the aging process. Here, we discuss that this concept integrates well with recent findings on cellular senescence, offering a novel view on the role of senescence in aging and age‐related disease. Cellular senescence has a well‐established role in cellular aging, but its impact on the rate of organismal aging is less defined. One of the most prominent features of cellular senescence is its association with macromolecular damage. The relationship between cell senescence and damage concerns both damage as a molecular signal of senescence induction and accelerated accumulation of damage in senescent cells. We describe the origin, regulatory mechanisms, and relevance of various damage forms in senescent cells. This view on senescent cells as carriers and inducers of damage puts new light on senescence, considering it as a significant contributor to the rise in organismal damage. Applying these ideas, we critically examine current evidence for a role of cellular senescence in aging and age‐related diseases. We also discuss the differential impact of longevity interventions on senescence burden and other types of age‐related damage. Finally, we propose a model on the role of aging‐related damage accumulation and the rate of aging observed upon senescent cell clearance.     

Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization

The intermediate chain of dynein forms a tight subcomplex with dimeric light chains LC8 and Tctex-1, and together they constitute the cargo attachment complex. There is considerable interest in identifying the role of these light chains in the assembly of the two copies of the intermediate chain. The N-terminal domain of the intermediate chain, IC1-289, contains the binding sites for the light chains, and is a highly disordered monomer but gains helical structure upon binding to light chains LC8 and Tctex-1. To provide insights into the structural and dynamic changes that occur in the intermediate chain upon light chains binding, we have used NMR spectroscopy to compare the properties of two distinct sub-domains of IC1-289: IC84-143 which is the light chains binding domain, and IC198-237, which contains a predicted coiled coil necessary for the increase in ordered structure upon light chain binding. Neither construct has stable secondary structure when probed by circular dichroism and amide chemical shift dispersion. Specific residues of IC84-143 involved in binding to the light chains were identified by their increase in resonance line broadening and the corresponding large intensity reduction in 1H-15N HSQC spectra. Interestingly, IC84-143 shows no sign of structure formation after binding to either LC8 or Tctex-1 or to both. IC198-237, on the other hand, contains a population of a nascent helix at low temperature as identified by heteronuclear NMR relaxation measurements, secondary chemical shifts, and sequential amide-amide connectivities. These data are consistent with a model for light chain binding coupled to intermediate chain dimerization through forming a coiled coil distant from the binding site.     

Solution structure of isoform 1 of Roadblock/LC7, a light chain in the dynein complex

Roadblock/LC7 is a member of a class of dynein light chains involved in regulating the function of the dynein complex. We have determined the three-dimensional structure of isoform 1 of the mouse Roadblock/LC7 cytoplasmic dynein light chain (robl1_mouse) by NMR spectroscopy. In contrast to a previously reported NMR structure of the human homolog with 96% sequence identity (PDB 1TGQ), which showed the protein as a monomer, our results indicate clearly that robl1 exists as a symmetric homodimer. The two beta3-strands pair with each other and form a continuous ten-stranded beta-sheet. The 25-residue alpha2-helix from one subunit packs antiparallel to that of the other subunit on the face of the beta-sheet. Zipper-like hydrophobic contacts between the two helices serve to stabilize the dimer. Through an NMR titration experiment, we localized the site on robl1_mouse that interacts with the 40 residue peptide spanning residues 243 through 282 of IC74-1_rat. These results provide physical evidence for a symmetrical interaction between dimeric robl1 and the two molecules of IC74-1 in the dynein complex.     

Assembly of a protein sharing epitopes with sperm dynein heavy chains into meiotic spindle in the prometaphase starfish oocyte

Starfish oocyte meiosis provides a good system for studying the mechanism for prometaphase chromosome movement. Since a protein sharing epitopes with sperm dynein might be a force generator for mitosis, the contribution of such a protein was assessed in this movement. Specific antibodies to heavy chains (HCs) and intermediate chains (ICs) of dynein subunits were affinity-purified from whole antidynein serum. We confirmed that the oocytes contain several polypeptides identical to sperm dynein subunits. The anti-HCs binding to in situ antigen was examined in the oocytes permeabilized with detergent at appropriate stages of maturation with special reference to tubulin and chromosomes, and the meiotic apparatus-establishing process was described in terms of a force generator (oocyte dynein). Before resumption of maturation, dynein HCs were particularly associated with prophase chromosomes within the germinal vesicle (GV). After GV breakdown, there was a striking local accumulation of dynein HCs in the "fading GV" (nuclear matrix). When chromosomes were pulled toward the central area between 2 asters, dynein was accumulated at first at the presumptive equator and then moved to the poles, showing uneven localization on the meiotic spindle.