The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.

/pdf/microtubule-polymerization-dynamics-27938flntt.pdf

Microtubule polymerization dynamics

Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. In addition, checkpoints respond to damage by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. Checkpoint loss results in genomic instability and has been implicated in the evolution of normal cells into cancer cells. Recent advances have revealed signal transduction pathways that transmit checkpoint signals in response to DNA damage, replication blocks, and spindle damage. Checkpoint pathways have components shared among all eukaryotes, underscoring the conservation of cell cycle regulatory machinery.

https://science.sciencemag.org/content/sci/274/5293/1664.full.pdf

Cell Cycle Checkpoints: Preventing an Identity Crisis

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity—by degrading CDK activators or inhibitors—and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

https://science.sciencemag.org/content/sci/274/5293/1652.full.pdf

How proteolysis drives the cell cycle

Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

Microtubules and actin filaments: dynamic targets for cancer chemotherapy

ANAPHASE and cytokinesis are key processes in the segregation of replicated chromosomes to the daughter cells: in anaphase, chromosomes move apart; in cytokinesis, a cleavage furrow forms midway between the separated chromosomes. Some evidence suggests that chromosomes may be involved both in controlling the timing of anaphase onset1–3 and in dictating the position of the cleavage furrow3. Other evidence indicates that the controlling mechanisms are intrinsic to the spindle and the cell4–7. Here we test these possibilities in grasshopper spermatocytes by observing spindles and cells after removal of chromosomes. We found that both anaphase and cytokinesis occur independently of chromosomes: stage-specific changes occur at an appropriate time and in the correct way, despite the absence of chromosomes. This finding is particularly noteworthy because chromosomes have an important impact on spindle microtubule assembly8,9 and the timing of anaphase onset10 in these cells.

'Anaphase' and cytokinesis in the absence of chromosomes

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video-enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.

/pdf/the-impact-of-chromosomes-and-centrosomes-on-spindle-1v5dvuls22.pdf

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

/pdf/chromosomes-initiate-spindle-assembly-upon-experimental-30r12ak5gp.pdf

Dahong Zhang

Papers

'Anaphase' and cytokinesis in the absence of chromosomes

The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.

Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes