Abstract
Microtubules (MTs) and associated proteins can self-organize into complex structures such as the bipolar spindle, a process in which RanGTP plays a major role. Addition of RanGTP to M-phase Xenopus egg extracts promotes the nucleation and self-organization of MTs into asters and bipolar-like structures in the absence of centrosomes or chromosomes. We show here that the complex proteome of these RanGTP-induced MT assemblies is similar to that of mitotic spindles. Using proteomic profiling we show that MT self-organization in the M-phase cytoplasm involves the non-linear and non-stoichiometric recruitment of proteins from specific functional groups. Our study provides for the first time a temporal understanding of the protein dynamics driving MT self-organization in M-phase.
Highlights
The ability to self-organize molecular machines and higher order complex forms is an essential feature of living systems [1, 2]
The mechanism at play involves the release of a class of Nuclear Localization Signal (NLS) containing proteins from inhibitory interactions with importins
These data indicate that RanGTP promotes a MT self-organization process that transits through intermediate steps involving changes in MT dynamics and organization
Summary
Xenopus Egg Extracts and RanGTP Dependent MTs—Xenopus laevis female and male frogs were purchased from Nasco and were used at an age between 1 and 3 years. The relative protein variation was obtained by calculating the difference of the average intensity of a given protein in a certain time point with the median of the protein intensity (from all the samples) resulting in a dynamics profile of each protein. The data for control and CBX3 silenced cells showed a normal distribution (D’Agostino & Pearson omnibus normality test) and equal variance (F test) In this case the statistical analysis was performed by unpaired two tailed t test. In the case of DnaJB6, cells were fixed in 4% PFA for 7 min after a preextraction step of 6 s (in BRB80 –1X, 0,5% triton and 1 mM DSP) and blocking and antibody dilution buffer was 0.5% BSA, 0.1% tritonX100 in PBS1X. For CBX3 transfer using semi-dry device, the transfer time should not be longer than 1 h from a 1.5 mm protein gel
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