A microtubule dynamics reconstitutional convention

305 The Rockefeller University Press $30.00 J. Cell Biol. Vol. 215 No. 3 305–307 https://doi.org/10.1083/jcb.201610066 Microtubules are cytoskeletal structures that serve as tracks for motor-based intracellular transport and underlie the organization of biological apparatuses, including the mitotic spindle, cilia, and the phragmoplast. In vivo, microtubules are highly dynamic and interconvert between phases of polymerization, pause, and depolymerization. In vitro, microtubules are dynamic, but their behavior poorly mimics that observed in living cells. Identifying the regulators responsible for tuning in vitro microtubule dynamics to match the in vivo behavior has been a quest in the reconstitution field. In a new study published in this issue, Moriwaki and Goshima have now identified the five key components that are necessary for the recapitulation of all three phases of microtubule dynamics in vitro. Nearly a century ago, mitotic spindle dynamics were observed. Determining the mechanisms that underlie these movements has remained a topic of active research. A key advance occurred in 1967 when Inoué and Sato (1967) used polarization microscopy to observe dynamic linear elements in the spindle. What were these linear elements made out of and how did they change their length? The discovery that colchicine disrupted the spindles positioned the field to use tritiated colchicine as a marker to identify tubulin, the protein formerly known as the colchicine binding protein (Borisy and Taylor, 1967). Tubulin is an obligate heterodimer consisting of α-tubulin and β-tubulin. The tubulin heterodimer polymerizes to form the microtubule lattice, mediated by both lateral and longitudinal interactions. The polarized nature of the tubulin heterodimer is propagated along the microtubule lattice, conferring the polymer with polarity: at one end, β-tubulin is exposed (the plus end), and at the other end, α-tubulin is exposed (the minus end). Each end of the polymer exhibits distinct dynamic behavior. Given that tubulin was identified as the microtubule building block, scientists should then be able to use purified tubulin to generate dynamic polymers that recapitulate the dynamic behavior observed in cells. Key steps toward achieving this goal required identifying the requisite nucleotide (GTP), divalent ion (magnesium), and optimal buffer conditions. In the mid-1980s, Mitchison and Kirschner (1984) polymerized tubulin in vitro, imaged fixed time points during the polymerization phase, and then diluted the system and imaged fixed time points during the depolymerization phase. They termed the nonequilibrium polymerization dynamics they observed “dynamic instability.” Under this process, microtubules do not reach a steady-state equilibrium length, but instead transition between phases of polymerization, pause, and depolymerization (Fig. 1 A). Underlying dynamic instability is the polymerization-dependent GTPase cycle of the tubulin heterodimer. Real-time visualization of dynamic instability came a few years later when dark field and superresolution video-enhanced differential interference contrast microscopy were used to observe microtubules transitioning between phases of polymerization and depolymerization (Horio and Hotani, 1986; Walker et al., 1988). Microtubule dynamics had been reconstituted, but there was one catch: the rates observed in vitro and the percentage of time microtubules spent in each phase (polymerization, depolymerization, and pause) did not correlate well with in vivo observations. Were there other factors required to regulate and tune dynamic instability? Enter the microtubule-associated proteins (MAPs). Two key MAP families were identified that promoted microtubule polymerization and depolymerization. In 1987, Gard and Kirschner (1987) purified XMAP215 and characterized its ability to potentiate microtubule polymerization. In 1999, Desai et al. (1999) identified Kinesin-13 family members as microtubule depolymerization factors. With these factors now in hand, the Hyman laboratory set out to reconstitute microtubule dynamics using purified tubulin, XMAP215, and Kinesin-13 (Kinoshita et al., 2001). With the inclusion of these MAPs, microtubule dynamics started to approach in vivo rates, but the limited sampling of the microtubule pause state in vitro suggested that yet another factor was required to stabilize microtubules in the pause state. Key steps forward included the identification of the CLIP-associating protein (CLA SP) family that promotes the microtubule pause state (Akhmanova et al., 2001; Sousa et al., 2007), as well as a microtubule plus end tracking complex involving EB1 and SLA IN2/Sentin that recruits XMAP215 and CLA SP to growing microtubule tips (van der Vaart et al., 2011; Li et al., 2012). With these molecular machines identified, the field was now positioned to test whether XMAP215, Kinesin-13, CLA SP, Sentin, and EB1 could collectively reproduce microtubule dynamics in vitro. Moriwaki and Goshima (2016) have now addressed this challenge. Given the extent to which these regulators had been characterized in Drosophila melanogaster cell culture, the authors decided to use purified In vitro reconstitution is the fundamental test for identification of the core components of a biological process. In this issue, Moriwaki and Goshima (2016. J. Cell Biol. https ://doi .org /10 .1083 /jcb .201604118) reconstitute all phases of microtubule dynamics through the inclusion of five key regulators and demonstrate that Polo kinase activity shifts the system from an interphase mode into an enhanced mitotic mode. A microtubule dynamics reconstitutional convention