Publications

2017
Remillard, D. ; Buckley, D. L. ; Paulk, J. ; Brien, G. L. ; Sonnett, M. ; Seo, H. S. ; Dastjerdi, S. ; Wühr, M. ; Dhe-Paganon, S. ; Armstrong, S. A. ; et al. Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands. Angewandte Chemie 2017, 56, 5738-5743.Abstract

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.

PDF icon 2017_remillard_bradner_angewandte.pdf
Presler, M. S. ; Van Itallie, E. ; Klein, A. M. ; Kunz, R. ; Coughlin, P. ; Peshkin, L. ; Gygi, S. ; Wühr, M. ; Kirschner, M. Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg. bioRxiv 2017, 145086.Abstract

Fertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

PDF icon 2017_presler_kirschner_biorxiv.pdf
Tassan, J. P. ; Wühr, M. ; Hatte, G. ; Kubiak, J. Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo. Results and problems in cell differentiation 2017, 61, 243-260.Abstract

Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

PDF icon 2017_tassan_kubiak_differentiation_and_cancer.pdf
Hasley, A. ; Chavez, S. ; Danilchik, M. ; Wühr, M. ; Pelegri, F. Vertebrate Embryonic Cleavage Pattern Determination. Advances in experimental medicine and biology 2017, 953, 117-171.Abstract

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

PDF icon 2017_hasley_pelegri_adv_exp_med_biol.pdf
Erickson, B. K. ; Rose, C. M. ; Braun, C. R. ; Erickson, A. R. ; Knott, J. ; McAlister, G. C. ; Wühr, M. ; Paulo, J. A. ; Everley, R. A. ; Gygi, S. P. A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization. Molecular Cell 2017.Abstract

Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.

PDF icon 2017_erickson_gygi_molecular_cell.pdf
2016
Pierre, A. ; Salle, J. ; Wühr, M. ; Minc, N. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos. Developmental Cell 2016, 39, 667-682.Abstract

Life for all animals starts with a precise 3D choreography of reductive divisions of the fertilized egg, known as cleavage patterns. These patterns exhibit conserved geometrical features and striking interspecies invariance within certain animal classes. To identify the generic rules that may govern these morphogenetic events, we developed a 3D-modeling framework that iteratively infers blastomere division positions and orientations, and consequent multicellular arrangements. From a minimal set of parameters, our model predicts detailed features of cleavage patterns in the embryos of fishes, amphibians, echinoderms, and ascidians, as well as the genetic and physical perturbations that alter these patterns. This framework demonstrates that a geometrical system based on length-dependent microtubule forces that probe blastomere shape and yolk gradients, biased by cortical polarity domains, may dictate division patterns and overall embryo morphogenesis. These studies thus unravel the default self-organization rules governing early embryogenesis and how they are altered by deterministic regulatory layers.

PDF icon 2016_pierre_minc_developmental_cell.pdf
Boke, E. ; Ruer, M. ; Wühr, M. ; Coughlin, M. ; Lemaitre, R. ; Gygi, S. P. ; Alberti, S. ; Drechsel, D. ; Hyman, A. A. ; Mitchison, T. J. Amyloid-like Self-Assembly of a Cellular Compartment. Cell 2016, 166, 637–650.Abstract

Most vertebrate oocytes contain a Balbiani body, a
large, non-membrane-bound compartment packed
with RNA, mitochondria, and other organelles. Little
is known about this compartment, though it specifies
germline identity in many non-mammalian vertebrates.
We show Xvelo, a disordered protein with
an N-terminal prion-like domain, is an abundant constituent
of Xenopus Balbiani bodies. Disruption of the
prion-like domain of Xvelo, or substitution with a
prion-like domain from an unrelated protein, interferes
with its incorporation into Balbiani bodies
in vivo. Recombinant Xvelo forms amyloid-like networks
in vitro. Amyloid-like assemblies of Xvelo recruit
both RNA and mitochondria in binding assays.
We propose that Xenopus Balbiani bodies form by
amyloid-like assembly of Xvelo, accompanied by
co-recruitment of mitochondria and RNA. Prion-like
domains are found in germ plasm organizing proteins
in other species, suggesting that Balbiani
body formation by amyloid-like assembly could be
a conserved mechanism that helps oocytes function
as long-lived germ cells.

PDF icon 2016_boke_mitchison_cell.pdf
2015
Braun, C. R. ; Bird, G. H. ; Wühr, M. ; Erickson, B. K. ; Rad, R. ; Walensky, L. D. ; Gygi, S. P. ; Haas, W. Generation of multiple reporter ions from a single isobaric reagent increases multiplexing capacity for quantitative proteomics. Anal Chem 2015, 87, 9855-63.Abstract

Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays.

PDF icon 2015_braun_gygi_analytical_chem.pdf
Wühr, M. ; Güttler, T. ; Peshkin, L. ; McAlister, G. C. ; Sonnett, M. ; Ishihara, K. ; Groen, A. C. ; Presler, M. ; Erickson, B. K. ; Mitchison, T. J. ; et al. The Nuclear Proteome of a Vertebrate. Curr Biol 2015, 25, 2663-71.Abstract

The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm; only ∼17% partition equally. A protein's native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1. This resulted in the expected re-localization of proteins toward the nucleus, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.

PDF icon 2015_wuhr_gygi_currbio.pdf
Mitchison, T. J. ; Ishihara, K. ; Nguyen, P. ; Wühr, M. Size Scaling of Microtubule Assemblies in Early Xenopus Embryos. Cold Spring Harb Perspect Biol 2015, 7 a019182.Abstract

The first 12 cleavage divisions in Xenopus embryos provide a natural experiment in size scaling, as cell radius decreases ∼16-fold with little change in biochemistry. Analyzing both natural cleavage and egg extract partitioned into droplets revealed that mitotic spindle size scales with cell size, with an upper limit in very large cells. We discuss spindle-size scaling in the small- and large-cell regimes with a focus on the "limiting-component" hypotheses. Zygotes and early blastomeres show a scaling mismatch between spindle and cell size. This problem is solved, we argue, by interphase asters that act to position the spindle and transport chromosomes to the center of daughter cells. These tasks are executed by the spindle in smaller cells. We end by discussing possible mechanisms that limit mitotic aster size and promote interphase aster growth to cell-spanning dimensions.

PDF icon 2015_mitchison_wuhr_csh_perspective.pdf
Peshkin, L. *; Wühr, M. *; Pearl, E. ; Haas, W. ; Freeman, R. M. ; Gerhart, J. C. ; Klein, A. M. ; Horb, M. ; Gygi, S. P. ; Kirschner, M. W. On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. Dev Cell 2015, 35, 383-94.Abstract

A biochemical explanation of development from the fertilized egg to the adult requires an understanding of the proteins and RNAs expressed over time during embryogenesis. We present a comprehensive characterization of protein and mRNA dynamics across early development in Xenopus. Surprisingly, we find that most protein levels change little and duplicated genes are expressed similarly. While the correlation between protein and mRNA levels is poor, a mass action kinetics model parameterized using protein synthesis and degradation rates regresses protein dynamics to RNA dynamics, corrected for initial protein concentration. This study provides detailed data for absolute levels of ∼10,000 proteins and ∼28,000 transcripts via a convenient web portal, a rich resource for developmental biologists. It underscores the lasting impact of maternal dowry, finds surprisingly few cases where degradation alone drives a change in protein level, and highlights the importance of transcription in shaping the dynamics of the embryonic proteome.

PDF icon 2015_peshkin_kirschner_dev_cell.pdf
Huttlin, E. L. ; Ting, L. ; Bruckner, R. J. ; Gebreab, F. ; Gygi, M. P. ; Szpyt, J. ; Tam, S. ; Zarraga, G. ; Colby, G. ; Baltier, K. ; et al. The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 2015, 162, 425-40.Abstract

Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.

PDF icon 2015_huttlin_gygi_cell.pdf
2014
Ishihara, K. ; Nguyen, P. A. ; Wühr, M. ; Groen, A. C. ; Field, C. M. ; Mitchison, T. J. Organization of early frog embryos by chemical waves emanating from centrosomes. Philos Trans R Soc Lond B Biol Sci 2014, 369.Abstract

The large cells in early vertebrate development face an extreme physical challenge in organizing their cytoplasm. For example, amphibian embryos have to divide cytoplasm that spans hundreds of micrometres every 30 min according to a precise geometry, a remarkable accomplishment given the extreme difference between molecular and cellular scales in this system. How do the biochemical reactions occurring at the molecular scale lead to this emergent behaviour of the cell as a whole? Based on recent findings, we propose that the centrosome plays a crucial role by initiating two autocatalytic reactions that travel across the large cytoplasm as chemical waves. Waves of mitotic entry and exit propagate out from centrosomes using the Cdk1 oscillator to coordinate the timing of cell division. Waves of microtubule-stimulated microtubule nucleation propagate out to assemble large asters that position spindles for the following mitosis and establish cleavage plane geometry. By initiating these chemical waves, the centrosome rapidly organizes the large cytoplasm during the short embryonic cell cycle, which would be impossible using more conventional mechanisms such as diffusion or nucleation by structural templating. Large embryo cells provide valuable insights to how cells control chemical waves, which may be a general principle for cytoplasmic organization.

PDF icon 2014_ishihara_mitchison_philtransb.pdf
Nguyen, P. A. ; Groen, A. C. ; Loose, M. ; Ishihara, K. ; Wühr, M. ; Field, C. M. ; Mitchison, T. J. Spatial organization of cytokinesis signaling reconstituted in a cell-free system. Science 2014, 346, 244-7.Abstract

During animal cell division, the cleavage furrow is positioned by microtubules that signal to the actin cortex at the cell midplane. We developed a cell-free system to recapitulate cytokinesis signaling using cytoplasmic extract from Xenopus eggs. Microtubules grew out as asters from artificial centrosomes and met to organize antiparallel overlap zones. These zones blocked the interpenetration of neighboring asters and recruited cytokinesis midzone proteins, including the chromosomal passenger complex (CPC) and centralspindlin. The CPC was transported to overlap zones, which required two motor proteins, Kif4A and a Kif20A paralog. Using supported lipid bilayers to mimic the plasma membrane, we observed the recruitment of cleavage furrow markers, including an active RhoA reporter, at microtubule overlaps. This system opens further approaches to understanding the biophysics of cytokinesis signaling.

PDF icon 2014_nguyen_mitchison_science.pdf
Wühr, M. ; Freeman, R. M. ; Presler, M. ; Horb, M. E. ; Peshkin, L. ; Gygi, S. P. ; Kirschner, M. W. Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database. Curr Biol 2014, 24, 1467-75.Abstract

BACKGROUND: Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes. RESULTS: Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis. CONCLUSION: To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates.

PDF icon 2014_wuhr_kirschner_currbiol.pdf
McAlister, G. C. ; Nusinow, D. P. ; Jedrychowski, M. P. ; Wühr, M. ; Huttlin, E. L. ; Erickson, B. K. ; Rad, R. ; Haas, W. ; Gygi, S. P. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 2014, 86, 7150-8.Abstract

Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8,378 proteins in union and 6,168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174,704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

PDF icon 2014_mcalister_gygi_analytical_chem.pdf
2012
Mitchison, T. ; Wühr, M. ; Nguyen, P. ; Ishihara, K. ; Groen, A. ; Field, C. M. Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells. Cytoskeleton (Hoboken) 2012, 69, 738-50.Abstract

Ray Rappaport spent many years studying microtubule asters, and how they induce cleavage furrows. Here, we review recent progress on aster structure and dynamics in zygotes and early blastomeres of Xenopus laevis and Zebrafish, where cells are extremely large. Mitotic and interphase asters differ markedly in size, and only interphase asters span the cell. Growth of interphase asters occurs by a mechanism that allows microtubule density at the aster periphery to remain approximately constant as radius increases. We discuss models for aster growth, and favor a branching nucleation process. Neighboring asters that grow into each other interact to block further growth at the shared boundary. We compare the morphology of interaction zones formed between pairs of asters that grow out from the poles of the same mitotic spindle (sister asters) and between pairs not related by mitosis (non-sister asters) that meet following polyspermic fertilization. We argue growing asters recognize each other by interaction between antiparallel microtubules at the mutual boundary, and discuss models for molecular organization of interaction zones. Finally, we discuss models for how asters, and the centrosomes within them, are positioned by dynein-mediated pulling forces so as to generate stereotyped cleavage patterns. Studying these problems in extremely large cells is starting to reveal how general principles of cell organization scale with cell size.

PDF icon 2012_mitchison_field_cytoskel.pdf
Wühr, M. ; Haas, W. ; McAlister, G. C. ; Peshkin, L. ; Rad, R. ; Kirschner, M. W. ; Gygi, S. P. Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster. Anal Chem 2012, 84, 9214-21.Abstract

Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion-ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low m/z reporter ions, the localization of these complement TMT (TMT(C)) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT(C) ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT(C) ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame.

PDF icon 2012_wuhr_gygi_analytical_chem.pdf
Nguyen, P. A. ; Ishihara, K. ; Wühr, M. ; Mitchison, T. J. Pronuclear migration: no attachment? No union, but a futile cycle!. Curr Biol 2012, 22, R409-11.Abstract

How do pronuclei migrate towards each other? The zebrafish futile cycle gene is shown to encode a maternally expressed membrane protein required for nuclear attachment and migration along the sperm aster.

PDF icon 2012_nguyen_mitchison_currbiol.pdf
2011
Field, C. M. ; Wühr, M. ; Anderson, G. A. ; Kueh, H. Y. ; Strickland, D. ; Mitchison, T. J. Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. J Cell Sci 2011, 124, 2086-95.Abstract

The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP-Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.

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