Spatial cues and not spindle pole maturation drive the asymmetry of astral microtubules between new and pre-existing spindle poles.

In many asymmetrically dividing cells, the microtubule-organizing centers (MTOCs; mammalian centrosome and yeast spindle pole body [SPB]) nucleate more astral microtubules on one of the two spindle poles than the other. This differential activity generally correlates with the age of MTOCs and contributes to orienting the mitotic spindle within the cell. The asymmetry might result from the two MTOCs being in distinctive maturation states. We investigated this model in budding yeast. Using fluorophores with different maturation kinetics to label the outer plaque components of the SPB, we found that the Cnm67 protein is mobile, whereas Spc72 is not. However, these two proteins were rapidly as abundant on both SPBs, indicating that SPBs mature more rapidly than anticipated. Superresolution microscopy confirmed this finding for Spc72 and for the.-tubulin complex. Moreover, astral microtubule number and length correlated with the subcellular localization of SPBs rather than their age. Kar9-dependent orientation of the spindle drove the differential activity of the SPBs in astral microtubule organization rather than intrinsic differences between the spindle poles. Together, our data establish that Kar9 and spatial cues, rather than the kinetics of SPB maturation, control the asymmetry of astral microtubule organization between the preexisting and new SPBs.