The mitotic spindle is constructed from microtubules (MTs) nucleated from centrosomes, chromosome proximal regions, and preexisting spindle MTs. Augmin, a recently identified protein complex, is a critical factor in spindle MT-based MT generation in Drosophila S2 cells. Previously, we identified one subunit of human augmin. Here, by using mass spectrometry, we identified the full human augmin complex of 8 subunits and show that it interacts with the ␥-tubulin ring complex (␥-TuRC). Unlike augmin-depleted S2 cells, in which the defect in spindle-mediated MT generation is mostly compensated by centrosomal MTs, augmin knockdown alone in HeLa cells triggers the spindle checkpoint, reduces tension on sister kinetochores, and severely impairs metaphase progression. Human augmin knockdown also reduces the number of central spindle MTs during anaphase and causes late-stage cytokinesis failure. A link between augmin and ␥-TuRC is likely critical for these functions, because a ␥-TuRC mutant that attenuates interaction with augmin does not restore function in vivo. These results demonstrate that MT generation mediated by augmin and ␥-TuRC is critical for chromosome segregation and cytokinesis in human cells.centrosome ͉ mitosis ͉ RNAi ͉ spindle checkpoint P roper segregation of sister chromatids during cell division relies on the assembly of a bipolar spindle during mitosis. Sister kinetochores associate with microtubules (MTs) from opposite poles in metaphase. When all of the kinetochores are attached to MTs and under tension, the spindle checkpoint is satisfied and the anaphase segregation of sister chromatids takes place (1, 2). Beginning at anaphase, spindle MTs reorganize to form a bundled and antiparallel MT structure between the segregating chromatids, a structure referred to as the central spindle.

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and g-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in g-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the g-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/g-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.