In all eukaryotes, chromosome segregation is accomplished by the mitotic spindle, a macromolecular machine composed of dynamically interacting proteins such as microtubules (MTs), molecular motors and microtubule-associated proteins (MAPs). The dynamic spindle was first observed by Shinya Inoue and Hidemi Sato in 1967 . Our current understanding of the mitotic spindle has exponentially increased through the use of wide-field epi-fluorescence imaging of MTs, made possible by fusions of green fluorescent protein to tubulin, and allowed for observation and manipulation of mitotic spindle in living cells. After decades of research and contribution from many researchers, the spindle is now viewed as a macromolecular machine that generates forces to assemble itself to segregate sister chromatids. Forces within the spindle are generated by kinesin and dynein, which are MT-based molecular motor proteins that generate directional movement along MTs and perform work. There are 45 kinesins spanning the fourteen kinesin families in human cells. Complimentarily, there are 16 dyneins spanning the nine families (two cytoplasmic and seven axonemal) in human cells. To date nine kinesin families and one cytoplasmic dynein are implicated in mitosis.
In addition to molecular motors, a plethora of MAPs contributes to mitotic spindle mechanics. Various MAPs function to 1) organize spindle pole, 2) organize and maintain the bipolar spindle, 3) attach MTs to kinetochores, and 4) help to move chromosomes. This large number of motors and MAPs creates complexity in dissecting core mechanisms of spindle dynamics. Spindle dynamics is apparent through all phases of mitosis. At prophase, the spindle is formed into a bipolar structure of defined length. At metaphase, spindle length remains relatively constant. The spindle structure contains 3 types of microtubules: astral, interpolar and kinetochore. At anaphase, kinetochore microtubules segregate chromosomes to the poles and then interpolar microtubules slide apart to extend the spindle length to further separate chromosomes.
In mammalian cells, during prophase chromosomes condense and the duplicated centrosomes are separated by kinesin-5 to form the bipolar spindle. Kinesin-12, kinesin-13, kinesin-14 and dynein localize to the poles where they function in spindle assembly and organization. Nuclear envelope breaks down at prometaphase, and kinesin-5 separates spindle poles apart and maintains bipolarity. In prometaphase, kinesin-4 and kinesin-10 localize to the chromosome arms and kinesin-7, kinesin-8, kinesin-13 and dynein to the kinetochore to facilitate microtubule capture and chromosome congression to the metaphase plate. Kinesin-8 also localizes to the kinetochore at metaphase to aid in chromosome congression and oscillation. During anaphase, sister chromatid separation and movement toward the poles are driven by kinesin-7 and kinesin-13 localized at the kinetochore and kinesin-12, kinesin-13, kinesin-14 localized at the poles. The spindle midzone, or central spindle, contains residual kinesin-5, kinesin-6 and kinesin-7. Dynein localizes to the cell cortex and pulls on astral microtubules to further separate the poles. During telophase, multiple kinesin families organize the spindle midzone. Mitosis in mammalian cells requires 9 kinesin families and a single dynein. Cartoon representation is modeled later.
Spindle Pole Body Duplication
In S. pombe, the spindle pole body (SPB), analogous to the centrosome in higher eukaryotes, is the principal microtubule organizing center (MTOC) during mitosis. SPBs are sites of MT nucleation and are essential for the formation of the mitotic spindle. The morphology of the SPB at different cell cycle stages and its mechanisms of duplication have been studied using static, fixed cells. Initially, the timing of SPB duplication has been judged by electron microscopy of cells synchronized with respect to cell cycle progression to be in G2. However, a recent study observed unduplicated SPBs in a number of wildtype G2 cells using electron tomography. In contrast, a report by Cande and colleagues has proposed that SPB duplication is initiated in G1/S by monitoring the morphology of SPBs in cells during cell cycle arrest/release experiments of fixed cells. Therefore, the timing of SPB duplication in the fission yeast cell cycle is controversial, partially because extensive studies using live cells are lacking. A recent report illustrates the power of using live-cell imaging in combination with electron microscopy to investigate SPB duplication and proposes that it initiates before G1/S phase and continues in G2 phase. The daughter SPB assembles at the tip of a mother SPB appendage called the bridge that maintains the duplicated SPBs associated until mitosis onset. The SPB bridge contains two major evolutionary conserved proteins, sfi1p and cdc31p/Centrin. Mutation in sfi1p or cdc31p leads to monopolar spindle validating that proper SPB duplication is essential for the formation of mitotic spindle.