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Full-Text Articles in Life Sciences
Fast Microtubule Dynamics In Meiotic Spindles Measured By Single Molecule Imaging: Evidence That The Spindle Environment Does Not Stabilize Microtubules, Thomas J. Maresca, D. J. Needleman, A. Groen, R. Ohi, L. Mirny, T. Mitchison
Fast Microtubule Dynamics In Meiotic Spindles Measured By Single Molecule Imaging: Evidence That The Spindle Environment Does Not Stabilize Microtubules, Thomas J. Maresca, D. J. Needleman, A. Groen, R. Ohi, L. Mirny, T. Mitchison
Thomas J. Maresca
Metaphase spindles are steady-state ensembles of microtubules that turn over rapidly and slide poleward in some systems. Since the discovery of dynamic instability in the mid-1980s, models for spindle morphogenesis have proposed that microtubules are stabilized by the spindle environment. We used single molecule imaging to measure tubulin turnover in spindles, and nonspindle assemblies, in Xenopus laevis egg extracts. We observed many events where tubulin molecules spend only a few seconds in polymer and thus are difficult to reconcile with standard models of polymerization dynamics. Our data can be quantitatively explained by a simple, phenomenological model—with only one adjustable parameter—in …
Welcome To A New Kind Of Tension: Translating Kinetochore Mechanics Into A Wait-Anaphase Signal, Thomas J. Maresca, E. D. Salmon
Welcome To A New Kind Of Tension: Translating Kinetochore Mechanics Into A Wait-Anaphase Signal, Thomas J. Maresca, E. D. Salmon
Thomas J. Maresca
Recent high-resolution studies of kinetochore structure have transformed the way researchers think about this crucial macro-molecular complex, which is essential for ensuring chromosome segregation occurs faithfully during cell division. Kinetochores mediate the interaction between chromosomes and the plus-ends of dynamic spindle microtubules and control the timing of anaphase onset by regulating the spindle assembly checkpoint (SAC). There is much debate in the SAC research community as to whether mitotic cells sense only microtubule attachment at the kinetochore, or both attachment and tension, before committing to anaphase. In this Commentary, we present a brief history of the tension-versus-attachment debate, summarize recent …
Intrakinetochore Stretch Is Associated With Changes In Kinetochore Phosphorylation And Spindle Assembly Checkpoint Activity, Thomas J. Maresca, E. D. Salmon
Intrakinetochore Stretch Is Associated With Changes In Kinetochore Phosphorylation And Spindle Assembly Checkpoint Activity, Thomas J. Maresca, E. D. Salmon
Thomas J. Maresca
Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing …
Functional Overlap Of Microtubule Assembly Factors In Chromatin-Promoted Spindle Assembly, Thomas J. Maresca, A. C. Groen, J. C. Gatlin, E. D. Salmon
Functional Overlap Of Microtubule Assembly Factors In Chromatin-Promoted Spindle Assembly, Thomas J. Maresca, A. C. Groen, J. C. Gatlin, E. D. Salmon
Thomas J. Maresca
Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, γ-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess …
Xenopus Tropicalis Egg Extracts Provide Insight Into Scaling Of The Mitotic Spindle, Thomas J. Maresca, M. D. Blower, K. S. Brown, T. C. Grammer, R. M. Harland, R. Heald
Xenopus Tropicalis Egg Extracts Provide Insight Into Scaling Of The Mitotic Spindle, Thomas J. Maresca, M. D. Blower, K. S. Brown, T. C. Grammer, R. M. Harland, R. Heald
Thomas J. Maresca
The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were …
Micromanipulation Studies Of Chromatin Fibers In Xenopus Egg Extracts Reveal Atp-Dependent Nucleosome Assembly Dynamics, Thomas J. Maresca, J. Yan, D. Skoko, R. Heald, J. F. Marko
Micromanipulation Studies Of Chromatin Fibers In Xenopus Egg Extracts Reveal Atp-Dependent Nucleosome Assembly Dynamics, Thomas J. Maresca, J. Yan, D. Skoko, R. Heald, J. F. Marko
Thomas J. Maresca
We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening–closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy …
Histone H1 Is Essential For Mitotic Chromosome Architecture And Segregation In Xenopus Laevis Egg Extracts, Thomas J. Maresca, J. Yan, D. Skoko, R. Heald, J. F. Marco
Histone H1 Is Essential For Mitotic Chromosome Architecture And Segregation In Xenopus Laevis Egg Extracts, Thomas J. Maresca, J. Yan, D. Skoko, R. Heald, J. F. Marco
Thomas J. Maresca
During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long …
The Condensin Complex Is Required For Proper Spindle Assembly And Chromosome Segregation In Xenopus Egg Extracts, Thomas J. Maresca, S. M. Wignall, R. Deehan, R. Heald
The Condensin Complex Is Required For Proper Spindle Assembly And Chromosome Segregation In Xenopus Egg Extracts, Thomas J. Maresca, S. M. Wignall, R. Deehan, R. Heald
Thomas J. Maresca
Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not …