(2014) Deep Proteomics of the Xenopus laevis Egg using an mRNA-Derived Reference Database. groups. Our study provides for the first time a temporal understanding of the protein dynamics driving MT self-organization in M-phase. MTs form patterns, but in combination with molecular motors they self-organize into defined structures such as asters, bundles, and networks (4C6). These basic properties may provide some principles underlying their self-organization into more complex cellular structures such as the bipolar spindle (1, 6, 7). Although, there is an Etimizol extensive list of spindle components obtained by proteomics (8C11), genetic screening (12, 13), and targeted functional studies, how all these components drive the strong self-assembly of the bipolar spindle is not fully understood yet. Moreover, it is unclear how all these components contribute or drive the assembly of such a dynamic structure. The egg extract system has been instrumental for understanding the molecular basis of M-phase MT assembly in the proximity of chromosomes and define a pathway essential for spindle assembly both in meiosis and mitosis (14). This pathway is usually triggered by the GTP bound form of the small GTPase Ran (15, 16). Indeed, the addition of RanGTP to M-phase egg extracts is sufficient to drive MT nucleation and business into asters and bipolar-like structures, called mini-spindles. The mechanism at play entails the release of a class of Nuclear Localization Transmission (NLS) made up of proteins from inhibitory interactions with importins. These so-called Spindle Assembly Factors (SAFs) have essential functions in RanGTP-dependent MT nucleation, stabilization, and business, and thereby in bipolar spindle assembly (17). Here, using mass spectrometry-based proteomics analysis we first validate the RanGTP-dependent MT assemblies as complex spindle-like structures. We then exploit this system to look for general principles driving MT and spindle self-organization. Our data suggest the non-linear recruitment of specific functional protein groups to MTs as they self-organize. EXPERIMENTAL PROCEDURES Xenopus Egg Extracts and RanGTP Dependent MTs female and male frogs were purchased from Nasco and were used at an age between 1 and 3 years. All experiments involving animals were performed according to standard protocols approved by the ethical committee of the Parc de Recerca Biomdica of Barcelona, Barcelona, Spain. New cytosolic-factor-arrested egg extracts (CSF extract) were prepared as previously explained (18) with the following modifications: Cytochalasin D was added to a final concentration of 13.3 g/ml (instead of 20 g/ml) and 1.5C2 ml of CSF extract were clarified by a second centrifugation step in 2 ml tubes for 15 min at 15,000 sperm nuclei as described (18) and MT aster and mini spindle assembly on addition of RanQ69LGTP. RanQ69LGTP Purification His-RanQ69L was expressed in BL21 (DE3) Rep4 E. Coli for 3 h at 30 C after induction with 2 mm IPTG. Cells were pelleted and washed using chilly PBS. Cells were lysed by incubation with Lysozyme (100 g/ml, 15 min at room temperature under constant stirring) in lysis buffer (PBS, 500 mm NaCl, 0.1 mm MgCl2, 0.5% Triton X-100, 1 mm DTT, 0.1 mm PMSF, 0.1 mm GTP). Cell lysates were cleared first by Etimizol centrifugation (30 min, 27,000 rcf at 4 C) and then by filtering using a low binding filter 0.45 m (Millex – HP Filter Unit, Fast Flow & Low Binding Millipore, Burlington, MA, ref SLHP033RS). The protein was purified by affinity chromatography using a 1 ml HisTRAP Etimizol HP Etimizol column (GE Healthcare, Chicago, IL, ref 17C5247-01) on FPLC-AKTA purifier. After incubation of the cell lysate, the column was washed with 10 column volumes of washing buffer (PBS, 500 mm NaCl, 0.1 mm MgCl2, 0.1 mm GTP, 40 mm Imidazole, pH Rabbit polyclonal to PNO1 7.7). Elution was performed by collecting 500 l fractions while mixing in gradually increasing concentrations of Elution buffer (PBS, 500 mm NaCl, 0.1 mm MgCl2, 0.1 mm GTP, 500 mm Imidazole) with Wash buffer. Fractions corresponding to the peak (around 150 mm Imidazole) were pooled Etimizol and dialyzed against CSF-XB. GTP was loaded onto RanQ69L by incubating the purified protein with 1 mm GTP for 2 h on ice. After incubation, 20 mm MgCl2 were added drop by drop to avoid precipitation of the protein. RanQ69LGTP was concentrated using Amicon Ultracel-3k membrane (Millipore, ref UFC900324) at the minimum concentration of 150 m. RanGTP Asters and Mini-spindle Assembly in Xenopus Egg Extracts RanQ69LGTP was.