Discrete States of a Protein Interaction Network Govern Interphase and Mitotic Microtubule Dynamics

The cytoplasm of eukaryotic cells is thought to adopt discrete “states” corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of “cytoplasmic states” regarding the microtubule system

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition

The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C[superscript CORT], a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C).National Institutes of Health (U.S.) (Grant GM39341

Widespread Changes in the Posttranscriptional Landscape at the Drosophila Oocyte-to-Embryo Transition

The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes that occur during this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity.National Institutes of Health (U.S.) (Grant GM39341

Discrete States of a Protein Interaction Network Govern Interphase and Mitotic Microtubule Dynamics

The cytoplasm of eukaryotic cells is thought to adopt discrete “states” corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of “cytoplasmic states” regarding the microtubule system

MT Stabilizing Activities of EB1 and XMAP215 in Interphase and Metaphase <i>Xenopus</i> Egg Extracts

<div><p>(A) Average size of asters in response to depletion of XMAP215 (ΔXMAP215) from the extract and the addition of recombinant EB1 to the ΔXMAP215 extract. Error bars represent the S.D. (<i>n</i> > 30). Upper panel: immunoblot (IB) of MAPs after indicated treatments.</p> <p>(B) Average size of asters in response to depletion of EB1 (ΔEB1) from the extract and the addition of recombinant XMAP215 to the ΔEB1 extract. Error bars represent the S.D. (<i>n</i> > 30). Upper panel shows the immunoblot (IB) of MAPs after the indicated treatments.</p> <p>(C) Still images of MT asters after the indicated treatments are shown in the upper panel. Table summarizing the dynamic parameters of MT growth in response to EB1 depletion (ΔEB1) and add back of recombinant EB1 into ΔEB1 interphase extracts is shown in the lower panel.</p></div

Validation of VIP Assay

<div><p>(A) VIP binding isotherm of EB1-EGFP to αEB1-antibody interaction fitted with a one site–binding model. A.U., arbitrary units.</p> <p>(B) Interaction of EB1-EGFP (prey: EGFP protein added to the extract) with APCMTBD (bait: recombinant protein added to the extract that binds to the anti-APC–coated beads) in metaphase (M, open bars) and interphase (I, filled bars). The three pairs of bars correspond to three independent experiments carried out in different extracts. A.U., arbitrary units; exp, experiment.</p> <p>(C) FRET binding isotherm of recombinant XMAP215-EGFP to recombinant EB1Cy3 in CSFXB buffer (left panel) and the VIP binding isotherm of recombinant XMAP215 to EB1-EGFP in CSFXB buffer (right panel). Both isotherms are fitted with one site–binding models. A.U., arbitrary units.</p> <p>(D) IP of XMAP215 or EB1 (left panel) and XMAP215/EB1-EGFP VIP (right panel) in interphase and metaphase extracts. Co-precipitation of EB1 or XMAP215 in IP experiments was probed by SDS-PAGE/immunoblotting with the respective antibodies (immunoblot [IB] with XMAP215 antibody and with EB1 antibody). In the VIP experiment, endogenous XMAP215 served as bait and EB1-EGFP as fluorescent prey. In the right panel, the four pairs of bars correspond to four independent experiments performed in metaphase (M, open bars) and interphase (I, filled bars) extracts. A.U., arbitrary units; exp, experiment.</p> <p>(E) Left panel shows IP pull down of 2 μM GST and GST-C-EB1 using apolyclonal αGST antibody. Co-precipitation of XMAP215 was probed by SDS-PAGE/immunoblotting with the XMAP215 antibody (IB: XMAP215). Immunoblot (IB) with monoclonal αGST antibody (m-GST) shows that GST and GST-C-EB1 were added to interphase and metaphase extracts accordingly. Right panel shows the VIP experiment of EB1-GST-C-EB1Cy5 and XMAP215-GST-C-EB1Cy5 in metaphase (M, open bars) and interphase (I, filled bars) extracts. Error bars represent the s.e.m. of VIP> derived from at least three different microscopic fields. A.U., arbitrary units, exp, experiment.</p></div

Change in the MAP Interaction Network at Interphase/Metaphase Transition

<div><p>(A) Pairwise interaction of MAPs in interphase and metaphase are represented as color-coded tables (upper panel). The color code corresponds to VIP> of at least three different microscopic fields. Proteins in green are EGFP tagged, and those in blue are Cy5 labeled. Below each table is a schematic representation of the network states in interphase and metaphase. Line thickness corresponds to interaction strength normalized to the strongest signal detected in interphase or metaphase. In the lower panel, an interphase aster is shown on the left and a mitotic one on the right</p> <p>(B) CDK1 activity induced a change in EB1-APCMTBD and EB1-XMAP215 interactions. EB1-EGFP fluorescence was measured simultaneously on beads coated with anti-XMAP215 and anti-APCMTBD antibodies in the presence of APCMTBD, and added to metaphase, interphase, and interphase extracts after addition of active, purified CDK1. Error bars represent the s.e.m. of vip> measured in six different microscopic fields. A.U., arbitrary units.</p> <p>(C) XMAP215/EB1-EGFP interaction in metaphase (open bars) and XMAP215/XKCM1-EGFP and APCMTBD/EB1-EGFP interaction in interphase (filled bars) before (control) and after incubation with 10 μM okadaic acid (OA). Error bars represent the s.e.m. of VIP> derived from at least three different microscopic fields.</p></div

The VIP Assay

<div><p>(A) Scheme of VIP methodology. AB1/AB2: specific antibodies recognizing endogenous bait.</p> <p>(B) Flow diagram of the automated image processing used for VIP.</p> <p>(C) Histograms of Alexa405 fluorescence (left panel) and EGFP fluorescence (right panel) on beads in an in vitro VIP experiment in which 1,600 nM EB1-EGFP was titrated into CSFXB buffer containing 300 nM XMAP215, anti-XMAP215 beads, and Alexa405-IgG beads. Different bead species (IgG/bait beads) are encoded by distinct Alexa405 emission (left panel). Each bead species is defined by the S.D. of Gaussian distributions (the black arrow marks the beginning and the grey arrow the end of the confidence interval) fitted to the Alexa405 histogram. Beads with Alexa405 intensities lying within the S.D. of the low-intensity population are defined as IgG beads. Beads with Alexa405 intensities lying within the S.D. of the high-intensity population are defined as bait beads (here: anti-XMAP215 beads). The emission of the two species (black: IgG beads; and red: anti-XMAP215 beads) in the EGFP channel is represented as a histogram (right panel). The VIP signal is calculated by subtracting the median intensity of bait beads in the EGFP channel by the median intensity of IgG beads in the EGFP channel only if there is a significant difference (<i>p</i> < 0.05, unpaired <i>t</i>-test) between these two populations.</p></div