Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerisation cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behaviour involving all three phases, by mixing tubulin with five Drosophila proteins, EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit. When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A, and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells, and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.