Amit Tzur

Bar Ilan University, Gan, Tel Aviv, Israel

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Publications (15)140.27 Total impact

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    ABSTRACT: Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. This analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos, a signature phenotype that could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
    Developmental biology. 08/2014;
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    ABSTRACT: Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.
    Journal of Structural Biology 03/2014; · 3.36 Impact Factor
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    ABSTRACT: Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH)(1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis.
    The EMBO Journal 02/2014; · 9.82 Impact Factor
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    ABSTRACT: The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and size-dependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.
    Proceedings of the National Academy of Sciences 09/2013; · 9.81 Impact Factor
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    ABSTRACT: Different types of mature B-cell lymphocytes are overall highly similar. Nevertheless, some B cells proliferate intensively, while others rarely do. Here, we demonstrate that a simple binary classification of gene expression in proliferating vs. resting B cells can identify, with remarkable selectivity, global in vivo regulators of the mammalian cell cycle, many of which are also post-translationally regulated by the APC/C E3 ligase. Consequently, we discover a novel regulatory network between the APC/C and the E2F transcription factors and discuss its potential impact on the G 1-S transition of the cell cycle. In addition, by focusing on genes whose expression inversely correlates with proliferation, we demonstrate the inherent ability of our approach to also identify in vivo regulators of cell differentiation, cell survival, and other antiproliferative processes. Relying on data sets of wt, non-transgenic animals, our approach can be applied to other cell lineages, as well as to human data sets.
    Cell cycle (Georgetown, Tex.) 08/2013; 12(18). · 5.24 Impact Factor
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    ABSTRACT: Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C complex, but not by the APC/C complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.
    PLoS ONE 01/2013; 8(2):e57532. · 3.53 Impact Factor
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    ABSTRACT: Cell cycle research greatly relies on synchronization of proliferating cells. However, effective synchronization of mammalian cells is commonly achieved by long exposure to one or more cell cycle blocking agents. These chemicals are, by definition, hazardous (some more than others), pose uneven cell cycle arrest, thus introducing unwanted variables. The challenge of synchronizing proliferating cells in G1 is even greater; this process typically involves the release of drug-arrested cells into the cycle that follows, a heterogeneous process that can truly limit synchronization. Moreover, drug-based synchronization decouples the cell cycle from cell growth in ways that are understudied and intolerable for those who investigate the relationship between these two processes. In this study we showed that cell size, as approximated by a single light-scatter parameter available in all standard sorters, can be used for synchronizing proliferating mammalian cells in G1 with minimal or no risk to either the cell cycle or cell growth. The power and selectivity of our method are demonstrated for human HEK293 cells that, despite their many advantages, are suboptimal for synchronization, let alone in G1. Our approach is readily available, simple, fast, and inexpensive; it is independent of any drugs or dyes, and nonhazardous. These properties are relevant for the study of the mammalian cell cycle, specifically in the context of G1 and cell growth.
    PLoS ONE 01/2013; 8(12):e83935. · 3.53 Impact Factor
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    ABSTRACT: We introduce a microfluidic system for simultaneously measuring single-cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 h of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.
    Nature Methods 08/2012; 9(9):910-2. · 23.57 Impact Factor
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    ABSTRACT: Currently, the reliable identification of peptides and proteins is only feasible when thoroughly annotated sequence databases are available. Although sequencing capacities continue to grow, many organisms remain without reliable, fully annotated reference genomes required for proteomic analyses. Standard database search algorithms fail to identify peptides that are not exactly contained in a protein database. De novo searches are generally hindered by their restricted reliability, and current error-tolerant search strategies are limited by global, heuristic tradeoffs between database and spectral information. We propose a Bayesian information criterion-driven error-tolerant peptide search (BICEPS) and offer an open source implementation based on this statistical criterion to automatically balance the information of each single spectrum and the database, while limiting the run time. We show that BICEPS performs as well as current database search algorithms when such algorithms are applied to sequenced organisms, whereas BICEPS only uses a remotely related organism database. For instance, we use a chicken instead of a human database corresponding to an evolutionary distance of more than 300 million years (International Chicken Genome Sequencing Consortium (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695-716). We demonstrate the successful application to cross-species proteomics with a 33% increase in the number of identified proteins for a filarial nematode sample of Litomosoides sigmodontis.
    Molecular &amp Cellular Proteomics 04/2012; 11(7):M111.014167. · 7.25 Impact Factor
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    ABSTRACT: Mitochondrial membrane potential provides a valuable indicator of cells' health and functional status. Cytometry- and microscopy-based analyses, in combination with fluorescent probes, are widely used to study mitochondrial behavior related to cellular pathways, most notably - apoptosis. The cyanine dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide) facilitates discrimination of energized and deenergized mitochondria because the normally green fluorescent dye forms red fluorescent aggregates when concentrated in energized mitochondria in response to their higher membrane potential. JC-1 fluorescence is usually excited by the 488 nm laser wavelength common in flow cytometers. In this study, we show that in practice this approach is not optimal for monitoring mitochondrial behavior. Investigation of fluorescence of JC-1 in solution and in cells using spectrofluorimetry, microscopy and flow cytometry reveals that excitation at 405 nm wavelength, now available on standard instruments, produces signals from aggregate fluorescence with considerably less spillover from dye monomer fluorescence than can be obtained using 488 nm excitation. The improved data are more accurate and eliminate the necessity for fluorescence compensation, making the use of the alternative excitation wavelengths beneficial for mitochondria-related biological and biomedial research.
    Cell Death & Disease 01/2012; 3:e430. · 6.04 Impact Factor
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    ABSTRACT: Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.
    PLoS ONE 01/2011; 6(1):e16053. · 3.53 Impact Factor
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    ABSTRACT: We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.
    Nature Methods 04/2010; 7(5):387-90. · 23.57 Impact Factor
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    ABSTRACT: The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.
    Proceedings of the National Academy of Sciences 01/2010; 107(4):1355-60. · 9.81 Impact Factor
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    ABSTRACT: A long-standing question in biology is whether there is an intrinsic mechanism for coordinating growth and the cell cycle in metazoan cells. We examined cell size distributions in populations of lymphoblasts and applied a mathematical analysis to calculate how growth rates vary with both cell size and the cell cycle. Our results show that growth rate is size-dependent throughout the cell cycle. After initial growth suppression, there is a rapid increase in growth rate during the G1 phase, followed by a period of constant exponential growth. The probability of cell division varies independently with cell size and cell age. We conclude that proliferating mammalian cells have an intrinsic mechanism that maintains cell size.
    Science 08/2009; 325(5937):167-71. · 31.20 Impact Factor

Publication Stats

272 Citations
140.27 Total Impact Points

Institutions

  • 2012–2014
    • Bar Ilan University
      • Faculty of Life Sciences
      Gan, Tel Aviv, Israel
  • 2010–2013
    • Massachusetts Institute of Technology
      • • Koch Institute for Integrative Cancer Research
      • • Department of Biological Engineering
      Cambridge, Massachusetts, United States
  • 2009–2012
    • Harvard Medical School
      • Department of Systems Biology
      Boston, Massachusetts, United States