1,103 research outputs found
Reconstructing Civility after Wrongdoing: A Place for Restorative Justice
Demonstration of mediation techniques that offer an alternative to retributive justice responses to wrongdoing
Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle
EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex
Roles of motor proteins in building microtubule-based structures: a basic principle of cellular design
AbstractEukaryotic cells must build a complex infrastructure of microtubules (MTs) and associated proteins to carry out a variety of functions. A growing body of evidence indicates that a major function of MT-associated motor proteins is to assemble and maintain this infrastructure. In this context, we examine the mechanisms utilized by motors to construct the arrays of MTs and associated proteins contained within the mitotic spindle, neuronal processes, and ciliary axonemes. We focus on the capacity of motors to drive the ‘sliding filament mechanism’ that is involved in the construction and maintenance of spindles, axons and dendrites, and on a type of particle transport called ‘intraflagellar transport’ which contributes to the assembly and maintenance of axonemes
Mitosis, microtubules, and the matrix
The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question
Three microtubule severing enzymes contribute to the “Pacman-flux” machinery that moves chromosomes
Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). We report that Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of γ-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing γ-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden our understanding of the molecular machinery used to move chromosomes
Sulfosuccinate and Sulfocarballylate Surfactants As Charge Control Additives in Nonpolar Solvents
A series
of eight sodium sulfonic acid surfactants with differently
branched tails (four double-chain sulfosuccinates and four triple-chain
sulfocarballylates) were studied as charging agents for sterically
stabilized poly(methyl methacrylate) (PMMA) latexes in dodecane. Tail
branching was found to have no significant effect on the electrophoretic
mobility of the latexes, but the number of tails was found to influence
the electrophoretic mobility. Triple-chain, sulfocarballylate surfactants
were found to be more effective. Several possible origins of this
observation were explored by comparing sodium dioctylsulfosuccinate
(AOT1) and sodium trioctylsulfocarballylate (TC1) using identical
approaches: the inverse micelle size, the propensity for ion dissociation,
the electrical conductivity, the electrokinetic or ζ potential,
and contrast-variation small-angle neutron scattering. The most likely
origin of the increased ability of TC1 to charge PMMA latexes is a
larger number of inverse micelles. These experiments demonstrate a
small molecular variation that can be made to influence the ability
of surfactants to charge particles in nonpolar solvents, and modifying
molecular structure is a promising approach to developing more effective
charging agents
Spindle microtubules in flux
Accurate and timely chromosome segregation is a task performed within meiotic and mitotic cells by a specialized force-generating structure - the spindle. This micromachine is constructed from numerous proteins, most notably the filamentous microtubules that form a structural framework for the spindle and also transmit forces through it. Poleward flux is an evolutionarily conserved mechanism used by spindle microtubules both to move chromosomes and to regulate spindle length. Recent studies have identified a microtubule-depolymerizing kinesin as a key force-generating component required for flux. On the basis of these findings, we propose a new model for flux powered by a microtubule-disassembly mechanism positioned at the spindle pole. In addition, we use the flux model to explain the results of spindle manipulation experiments to illustrate the importance of flux for proper chromosome positioning
Microtubule binding by dynactin is required for microtubule organization but not cargo transport
Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150glued, binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150glued, we replaced the wild-type p150glued in Drosophila melanogaster S2 cells with mutant ΔN-p150 lacking residues 1–200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150glued with ΔN-p150glued has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150glued has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo
Centrosome loss results in an unstable genome and malignant prostate tumors
Localized, nonindolent prostate cancer (PCa) is characterized by large-scale genomic rearrangements, aneuploidy, chromothripsis, and other forms of chromosomal instability (CIN), yet how this occurs remains unclear. A well-established mechanism of CIN is the overproduction of centrosomes, which promotes tumorigenesis in various mouse models. Therefore, we developed a single-cell assay for quantifying centrosomes in human prostate tissue. Surprisingly, centrosome loss-which has not been described in human cancer-was associated with PCa progression. By chemically or genetically inducing centrosome loss in nontumorigenic prostate epithelial cells, mitotic errors ensued, producing aneuploid, and multinucleated cells. Strikingly, transient or chronic centrosome loss transformed prostate epithelial cells, which produced highly proliferative and poorly differentiated malignant tumors in mice. Our findings suggest that centrosome loss could create a cellular crisis with oncogenic potential in prostate epithelial cells.6 month embargo; published online: 2 September 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]
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