Salmon Edward D.

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Salmon

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Edward D.

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Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly

(American Society for Cell Biology, 2009-04-15) Groen, Aaron C. ; Maresca, Thomas J. ; Gatlin, Jesse C. ; Salmon, Edward D. ; Mitchison, Timothy J.

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, {gamma}-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 XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either {gamma}-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.
Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

(Rockefeller University Press, 2009-02-03) Maresca, Thomas J. ; Salmon, Edward D.

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 centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.
Microtubule plus-end dynamics in Xenopus egg extract spindles

(American Society for Cell Biology, 2004-02-06) Tirnauer, Jennifer S. ; Salmon, Edward D. ; Mitchison, Timothy J.

Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at ~11 µm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization (~30 µm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by {alpha}-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.
Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles

(American Society for Cell Biology, 2005-03-23) Mitchison, Timothy J. ; Maddox, P. ; Gaetz, J. ; Groen, Aaron C. ; Shirasu, M. ; Desai, Ankur R. ; Salmon, Edward D. ; Kapoor, Tarun M.

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.
Bipolarization and poleward flux correlate during xenopus extract spindle assembly

(American Society for Cell Biology, 2004-09-22) Mitchison, Timothy J. ; Maddox, P. ; Groen, Aaron C. ; Cameron, Lisa ; Perlman, Z. ; Ohi, Ryoma ; Desai, Ankur R. ; Salmon, Edward D. ; Kapoor, Tarun M.

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.
Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity

( 2009-05) Maresca, Thomas J. ; Groen, Aaron C. ; Gatlin, Jesse C. ; Ohi, Ryoma ; Mitchison, Timothy J. ; Salmon, Edward D.

During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.
Directly probing the mechanical properties of the spindle and its matrix

(Rockefeller University Press, 2010-02-22) Gatlin, Jesse C. ; Matov, Alexandre ; Danuser, Gaudenz ; Mitchison, Timothy J. ; Salmon, Edward D.

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.
Chromosomes can congress to the metaphase plate before biorientation

( 2005-12-09) Kapoor, Tarun M. ; Lampson, Michael A. ; Hergert, Polla ; Cameron, Lisa ; Cimini, Daniela ; Salmon, Edward D. ; McEwen, Bruce F. ; Khodjakov, Alexey

The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle (i.e., “bi-oriented”). We found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy and correlative electron microscopy, we found that mono-oriented chromosomes could glide towards the spindle equator alongside kinetochore fibers attached to other already bi-oriented chromosomes. This congression mechanism depended on the kinetochore-associated plus enddirected microtubule motor CENP-E (kinesin-7).

Salmon Edward D.

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