Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize for the first time complete 3-D movements of individual kinetochores throughout mitosis in non-transformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected new details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high-density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.

Summary The centrosome is the principal microtubule organizing center (MTOC) of animal cells [1]. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as that of the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP [2] is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormal long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.