Current Biology, Vol. 14, R797–R805, September 21, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.09.021 Mechanisms and Molecules of the Review Mitotic Spindle Sharat Gadde and Rebecca Heald* Microtubule nucleating sites exert a major influence on spindle assembly. Most animal cells contain a single microtubule nucleating structure, the centro- In all eukaryotes, morphogenesis of the microtubule some, which consists of a pair of centrioles sur- cytoskeleton into a bipolar spindle is required for the rounded by amorphous material that harbors faithful transmission of the genome to the two templates for microtubule nucleation. The polarity of daughter cells during division. This process is facili- microtubule growth from centrosomes, with their tated by the intrinsic polarity and dynamic properties minus-ends tethered and their plus-ends extending of microtubules and involves many proteins that outward, facilitates proper organization of the spindle. modulate microtubule organization and stability. How is the spindle set up? By the onset of mitosis, Recent work has begun to uncover the molecular at prophase, the centrosome and the chromosomes mechanisms behind these dynamic events. Here we have duplicated and a cascade of events occurs, describe current models and discuss some of the including nuclear envelope breakdown, chromosome complex repertoire of factors required for spindle condensation and centrosome separation (Figure 1B). assembly and chromosome segregation. An increase in the frequency of microtubule shrinkage events, called catastrophes [2], and a decrease in events rescuing growth [3] contribute to the disman- tling of the interphase array, thus allowing interaction Introduction between dynamic microtubule plus-ends and the con- Essential to the process of cell division is the mitotic densed chromosomes. During prometaphase, some spindle, which partitions a complete set of chromo- microtubules emanating from one centrosome attach somes to each daughter cell. The spindle consists of to the kinetochore of one of the duplicated chro- microtubules, polar dynamic fibers that polymerize matids. Subsequent attachment of the sister kineto- from tubulin subunits, as well as hundreds of other chore to microtubules growing from the other proteins that function together to orchestrate chromo- centrosome results in the bi-orientation of the chro- some segregation. These include a large set of micro- mosome and its eventual congression to the center of tubule-based motor proteins that use ATP hydrolysis the antiparallel microtubule array. Once all of the chro- to generate movement, or alter microtubule dynamics. mosomes are bi-oriented and aligned, the cell is in While the basic steps of spindle assembly and metaphase. In addition to the kinetochore fibers, other anaphase chromosome segregation have been docu- populations of microtubules also contribute to the mented since the emergence of light microscopy bipolar structure, including the interpolar microtubules (Figure 1), pioneering techniques have continued to that overlap to form an antiparallel array, and the tell us new things about spindle microtubule dynam- astral microtubules, that extend from each centro- ics. Molecular approaches, empowered by complete some away from the spindle where they can interact genome sequences, are continuing to identify the pro- with the cell cortex (Figure 1A). teins responsible for the phenomena observed. In this When the chromosomes are aligned and oriented, a review, we highlight some of the latest techniques cellular checkpoint is satisfied, and anaphase A ensues developed and molecules identified that shed light on as sister chromosomes separate and move toward how the spindle assembles and functions to segregate opposite spindle poles with their kinetochores leading chromosomes. (Figure 1B). Anaphase B also contributes to chromo- some segregation, as spindle poles separate and the Spindle Anatomy and Steps of Assembly central spindle forms. Telophase marks the reformation Organizing a specific arrangement of microtubules of the nuclear envelopes around daughter cell nuclei as and chromosomes within the spindle is central to how the cytokinetic furrow pinches the cell into two. the process works (Figure 1A). Microtubules must be Although memorizing of the phases of mitosis has arranged into a bipolar array, such that each half tortured students for decades, understanding how spindle contains uniformly oriented microtubules, with these events actually occur continues to occupy cell their minus-ends at the pole and their plus-ends biologists, as a complete molecular model has yet to extending away. Each duplicated chromosome has a be obtained. Although Figure 1 is reasonably accurate pair of specialized structures at its centromere, called in depicting a static view of progression through kinetochores, which function to attach sister chro- mitosis, it does not convey the dynamic nature of the matids to microtubules from opposite spindle poles, spindle. Furthermore, the canonical diagram does not to allow for directed translocation of chromosomes take into account the exceptions to the rules, which within the spindle [1]. have been extremely instructive in elucidating the principles underlying spindle assembly. Below we Department of Molecular and Cell Biology, University of describe some models of spindle dynamics, and then California, Berkeley, Berkeley, California 94720-3200, USA. launch into a description of the molecules that under- *E-mail: [email protected] lie the behaviors seen. Review R798 Figure 1. Spindle anatomy and the cell A cycle. Kinetochore (A) Features of the metaphase mitotic Centrosome spindle. With their minus ends tethered at the spindle poles, microtubules extend Kinetochore either to the kinetochores of paired chro- fiber matids (kinetochore fibers), to the central spindle where they form an overlapping Interpolar antiparallel array (interpolar micro- microtubules tubules), or away from the spindle towards the cell cortex (astral micro- tubules). (B) The stages of mitosis illus- trating microtubule reorganization and chromosome translocation. During inter- phase, the chromosomes and centro- some are replicated. At prophase, chromosome condensation begins, cen- Astral trosomes separate and the nuclear enve- microtubule Paired lope breaks down. During prometaphase, chromatids chromosomes are captured by micro- tubules growing from the separated cen- B trosomes and bi-orient, congressing to the center of the spindle at metaphase. Anaphase marks the loss of cohesion between sister chromatids and their movement to opposite spindle poles, which move apart to further separate daughter nuclei re-forming in telophase (not shown) prior to cytokinesis and the Interphase Prophase return to interphase. • centrosome duplication • nuclear envelope breakdown • DNA replication • chromosome condensation • cell growth • centrosome separation Prometaphase • chromosome capture • chromosome congression Anaphase • chromatid segregation • spindle pole separation Metaphase • chromosomes aligned Current Biology Multiple Mechanisms at Work (Figure 2B) [5], was not thought to apply to somatic One of the ‘special’ cases that has shed light on the cells harboring centrosomes. However, several lines of process is the assembly of the female meiotic spindle, evidence have changed this view. which occurs in the absence of centrosomes. Originally A major argument that self-organization is at work, thought to be an anomaly, the mechanisms by which a even in the presence of centrosomes, is that spindle bipolar microtubule array forms in this situation are now assembly can proceed after centrosome function has believed to be a general feature of spindle assembly. been abolished. For example, mutations have been The predominant model of spindle assembly in the identified in Drosophila that inactivate centrosomes, presence of centrosomes is based on microtubule yet functional spindles still form [6–8], as they do in a dynamic instability and is known as the “search-and- related insect, Sciara, which can produce capture” model [4]: Microtubules emanating from a parthenogenic embryos lacking centrosomes [9]. centrosome undergo cycles of growth and shrinkage, When the centrosome is physically removed in verte- randomly probing the cytoplasm until running into a brate somatic cells using a laser beam or micro- kinetochore, with which they form a stable attachment surgery, functional bipolar spindles form nevertheless (Figure 2A). Because microtubules from duplicated cen- [10–12]. The major effect on mitotic progression in the trosomes encounter bivalent kinetochores, a bipolar absence of centrosomes is that spindles are more spindle forms. In contrast, in the absence of centro- often misoriented due to the loss of astral micro- somes, microtubules polymerize in a disorganized tubules, which can decrease the fidelity of cytokinesis. fashion without focal nucleation sites and yet a spindle More evidence that mechanisms in addition to forms. Motor-dependent mechanisms must be invoked search-and-capture are at work comes from experi- to sort these randomly oriented microtubules into a ments showing that spindles can form in the absence bipolar structure. The ‘self-organization’ model based of kinetochores, or even chromosomes. One system on observations of acentrosomal spindle assembly that has been particularly useful to directly compare Current Biology R799 Figure 2. Models of spindle assembly. (A) ‘Search-and-Capture’: