Biomedical subjects

J C Waters

Publications and source records attributed to J C Waters.

10 recordsLinked to original sources

PubMed2001

Checkpoint signals in grasshopper meiosis are sensitive to microtubule attachment, but tension is still essential.

The spindle checkpoint detects errors in kinetochore attachment to microtubules and delays anaphase if attachment is improper. The checkpoint is activated by attachment-sensitive components including Mad2 and certain phosphorylated proteins detected by the 3F3/2 antibody. We have studied Mad2 and 3F3/2 immunofluorescence in grasshopper spermatocytes. As in other cells, unattached kinetochores are loaded with Mad2 and are highly phosphorylated, whereas after proper attachment, Mad2 is lost and kinetochores are dephosphorylated. What is it about proper attachment that produces these changes--is it microtubule attachment itself or is it the tension from mitotic forces that follows proper attachment? Using micromanipulation, we created an intermediate state, weak attachment, that provides an answer. Weakly attached kinetochores are not under tension and have few kinetochore microtubules. Despite the absence of tension, many weakly attached kinetochores lose their Mad2 and become dephosphorylated. Therefore we conclude that microtubule attachment determines both Mad2 binding and phosphorylation. Nevertheless, tension plays an absolutely essential role. Tension elevates the number of kinetochore microtubules to the level necessary for the complete loss of Mad2 and dephosphorylation from all kinetochores. This gives a reliable 'all clear' signal to the checkpoint, allowing the cell to progress to anaphase.

Animals↗

PubMed1999

Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis.

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.

Animals↗

PubMed1998

Localization of Mad2 to kinetochores depends on microtubule attachment, not tension.

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301-1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242-246; Li, Y., and R. Benezra. Science. 274: 246-248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK1 cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929-939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK1 cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after approximately 12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.

Animals↗

PubMed1997

Pathways of spindle assembly.

Recent studies have revealed that, in some systems, chromatin has the ability to stabilize microtubules and organize them into bipolar spindles independently of kinetochores and centrosomes. In addition, several molecules have been identified recently that are necessary for spindle assembly; these include proteins that regulate microtubule dynamics, proteins that organize microtubule minus ends into spindle poles, and members of the kinesin superfamily that reside on the chromosome arms.

Animals↗

PubMed1996

Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores.

The spindle assembly checkpoint delays anaphase until all chromosomes are attached to a mitotic spindle. The mad (mitotic arrest-deficient) and bub (budding uninhibited by benzimidazole) mutants of budding yeast lack this checkpoint and fail to arrest the cell cycle when microtubules are depolymerized. A frog homolog of MAD2 (XMAD2) was isolated and found to play an essential role in the spindle assembly checkpoint in frog egg extracts. XMAD2 protein associated with unattached kinetochores in prometaphase and in nocodazole-treated cells and disappeared from kinetochores at metaphase in untreated cells, suggesting that XMAD2 plays a role in the activation of the checkpoint by unattached kinetochores. This study furthers understanding of the mechanism of cell cycle checkpoints in metazoa and provides a marker for studying the role of the spindle assembly checkpoint in the genetic instability of tumors.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1996

Cytoskeleton: a catastrophic kinesin.

The 'plus' ends of microtubules exhibit dynamic instability, switching stochastically from growth to shortening phases. The first endogenous regulator of such 'catastrophes' has been identified, and is a kinesin-related microtubule motor protein.

Animals↗

PubMed1996

The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.

During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.

Anaphase↗

PubMed1996

Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push.

Experimentally introduced tension on kinetochores and their centromeres has been shown to stabilize kinetochore attachment to microtubules, modify kinetochore directional instability, and regulate cell-cycle progression into anaphase. In mitosis, kinetochore tension and the stretch of centromere chromatin are produced by the movement of sister kinetochores toward opposite poles and astral ejection forces on the chromosome arms. However, newt lung cell kinetochores oscillate between poleward and away from the pole motility states throughout mitosis, indicating kinetochores are not under constant tension. To test whether kinetochores are under net tension while they are oscillating, and how often they are under compression and pushing into the chromosome, we measured the distance between sister kinetochores in newt lung cells using both video-enhanced differential interference contrast microscopy (VE-DIC) and immunofluorescence microscopy. We found that for chromosomes in which sister kinetochores are attached to opposite spindle poles, centromeres are, on average, stretched (2.2 microns in living cells and 1.8 microns in fixed cells) with respect to the inter-kinetochore 'rest' length (1.1 microns in living and fixed cells). For chromosomes in which only one kinetochore is attached to the spindle, the centromere chromatin associated with the tethered kinetochore is, on average, stretched to approximately half of the average inter-kinetochore distance measured for chromosomes in which both kinetochores are attached. We conclude that while newt lung cell kinetochores oscillate between states of P and AP movement, they are under tension approximately 90% of the time and under compression less than 6% of the time.

Animals↗

PubMed1995

Chromosomes take an active role in spindle assembly.

The assembly of a bipolar spindle is essential for the accurate segregation of replicated chromosomes during cell division. Do chromosomes rely solely on other cellular components to regulate the assembly of the bipolar spindle or are they masters of their own fate? In the Zhang and Nicklas(1) study reviewed here, micromanipulation techniques and video microscopy were used to demonstrate the different roles that chromosome arms, kinetochores and centrosomes play in bipolar spindle assembly.

Animals↗

PubMed1993

The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster.

A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.

Anaphase↗