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We next sought to determine how these factors influence the kinetics of microtubule nucleation. An individual microtubule is marked by a single GFP-EB1 spot at its growing plus tip, and thus, counting the number of EB1-GFP spots using an automated algorithm provides a direct measure of microtubule number over time. Application of this method to TIRF imaging only counts microtubules [http://www.selleckchem.com/products/sch772984.html SCH772984 solubility dmso] near the coverslip, but nonquantitative confocal and widefield imaging performed on similar reactions (not shown) suggested this reflects the total population. In the wild-type extract, microtubule numbers increased very slowly over time (Figure?2). Addition of RanQ69L dramatically stimulated the appearance of microtubules compared to the control extract; the kinetics revealed an initial lag phase followed by a sigmoidal increase in microtubule number. This result is in general agreement with experiments by Ribbeck and Clausen (Clausen and Ribbeck, 2007) who measured total microtubule mass after RanQ69L addition. Addition of TPX2 with RanQ69L further accelerated the rate of formation, as well as the number of microtubules, compared to RanQ69L alone. We next examined the [http://www.selleckchem.com/products/Bortezomib.html Bortezomib datasheet] angles of daughter filaments that emerge from the mother microtubule; a branch angle of zero degrees is defined as a parallel growth and 180�� as antiparallel growth (Figure?3A). The branch angle was shallow in most instances, with the daughter microtubule growing parallel or nearly parallel to the template microtubule ([https://en.wikipedia.org/wiki/Amrinone Amrinone] addition (Figure?3A; Movies S1, S2, and S3), thus replicating the polarity of parent microtubules in the emerging network. Microtubule nucleation occurred predominantly along the length of the original microtubule (?60%) and at the very distal minus end (?30% of events, Figure?3B). Many nucleation events along the length of the microtubule occurred at the same site (within the resolution of the microscope) of a previous nucleation event (?33%). In rare cases, a growing EB1 comet appeared to split apart, thereby generating two new microtubules from a growing plus end. Using the robust branching microtubule nucleation assay in the presence of both RanQ69L and TPX2, we tested for the involvement of additional factors by immunodepletion. Formation of fan-shaped, branching microtubule structures that originated by branching microtubule nucleation was not affected by a control immunodepletion with nonspecific IgG antibodies (Figure?4A; Movie S6; 1.42 �� 0.56 fans [defined as a branching cluster of more than five MTs] per 54?�� 54?��m field of view; mean �� SEM of two independent experiments with ?50 fields each).