Amit Tzur

Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation

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/CCDH1‐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.

Overcoming Species Boundaries in Peptide Identification with Bayesian Information Criterion-driven Error-tolerant Peptide Search (BICEPS)

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.

Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the \(APC/C^{Cdh1}\) Complex

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/CCdh1 complex, but not by the APC/CCdc20 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.

Cytokinetic abscission is an acute G1 event

Animal cell division ends with the cutting of the microtubule and membrane intercellular bridge connecting the 2 daughter cells. This process, known as cytokinetic abscission (abscission), is widely regarded as the last step of cytokinesis, i.e., the last step of the cell cycle. Major breakthroughs have been recently achieved, illuminating mechanistic aspects of abscission; however, the timing of abscission with respect to the mammalian cell cycle remains unclear. In this study, we carefully measured the onset and progression of abscission in dividing cells expressing a G1 reporter. We conclude that abscission commences long after cells enter the G1 phase. Affiliating abscission with G1 is beyond semantics since it essentially postulates that the last step of the cell cycle is regulated in, and probably by, the following cycle.

Crystal structure of the extracellular juxtamembrane region of Robo1.

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.

Using standard optical flow cytometry for synchronizing proliferating cells in the G1 phase.

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.

The interface of nanoparticles with proliferating mammalian cells

To the Editor — Cell cycle and cell growth (that is, increase of cell size) are distinct processes that are coupled in proliferating cells. Together, these processes ensure genomic and size stability essential for proper cell reproduction and function1–3. Proliferating cells grow continuously from birth to division, doubling their mass and volume (two common metrics of cell size) during their life cycle (Fig. 1a). The process of cytokinesis generates daughter cells that are approximately half the size of the mother cell4–6. Although proliferating cells progress through the cell cycle at different paces and grow at different rates, G1 cells are on average smaller than S-phase cells, which, in turn, are smaller than cells in G2 or mitosis4–7. The coupling between cell volume and cell-cycle progression can be manifested by standard optical cytometry, as is shown here for A549 and L1210 cells (Fig. 1b and Supplementary Fig. 1). Here, information on cell volume is obtained either from light scattering (side-scatter pulse area, SSC-A) or postsort by the Coulter principle (Fig. 1b and Supplementary Fig. 2; refs 7,8). Information on the cell cycle is obtained by DNA quantification. An in-depth understanding of the interface of nanoparticles (NPs) with proliferating cells can illuminate facets of cell physiology relevant to the nanomedicine of human diseases associated with malignant proliferation. A recent study suggested a role for the cell cycle in NP uptake by proliferating mammalian cells9. This study was based mainly on long-term (24-h) exposure of A549 human epithelial cells to 40-nm carboxylate-modified polystyrene (PS-COOH) microspheres. The concentration of these NPs in proliferating cells was reported to be highest in the G2/M phase of the cell cycle, followed by the S and the G1 phases, even though cells in different phases of the cell cycle were found to internalize NPs at similar rates. This study, however, did not distinguish between cell cycle and cell growth and was performed without taking into account cell volume. In fact, the cellular NP concentrations, although specified as cell-cycle-dependent, were not measured. We have long been interested in the interplay between cell cycle, cell growth and cell size4–8,10, and decided to investigate the behaviour of NPs in this context. Our first goal was to characterize the relationship between cellular amounts of NPs (determined by fluorescence intensity) and cell volume (estimated by SSC-A) in adherent A549 cells and in unattached L1210 cells. The latter cell type is optimal for cytometry-based analyses8,10. We incubated cells with 40-nm PS-COOH modified fluorescent microspheres at 25 μg ml–1 (from here on simply referred to as NPs). This is a non-saturating dose at which virtually all cells internalize NPs (Supplementary Fig. 3) without a significant impact on cell volume and SSC-A distributions, and the quality of cell volume approximation by SSC-A (Supplementary Fig. 4). We studied live cells, avoiding fixation-driven distortion of cellular metrics. In both cell types, we detected a strong positive correlation between cellular amounts of NPs and SSC-A after 24-h incubation (Fig. 1c). This correlation was observed for the whole SSC-A range, that is, throughout the life cycle of all cells within the examined population. Similar results were obtained for various incubation times (Supplementary Fig. 5) and for primary cells (Supplementary Fig. 6). L1210 cells are nearly spherical. Thus, their volume can be calculated from cell area or perimeter under spherical assumption6,10, allowing direct measurement of both NP amount and concentration by microscopy. L1210 cells expressing a membrane green fluorescent protein (GFP) marker simplifies geometric measurements (Fig. 1d and Supplementary Fig. 7, and ref. 6). As shown in Fig. 1d, a nearly perfect linear correlation between cellular NP amount and cell volume was observed after 24-h incubation. These results effectively demonstrate that the concentration of NPs in proliferating L1210 cells is approximately constant. Similar results were obtained following incubation with higher, non-saturating, doses of NPs (Supplementary Fig. 8). In both A549 and L1210 cells, the positive correlation between cellular NPs and cell volume, approximated by SSC-A, was unrelated to a particular phase of the cell cycle (Fig. 1e and Supplementary Fig. 9). We also examined the NP/SSC-A relationship in cell-cycle-arrested L1210 cells, and in resting (G0) A549 cells (Fig. 1f–i and Supplementary Fig. 10). This allowed us to examine NPs when the cell cycle is decoupled from cell growth and size (Fig. 1f,g) or irrespective of the cell cycle (Fig. 1h,i). The amount of cellular NPs in L1210 cells arrested in S-phase or prometaphase and in resting A549 cells was found to be proportional to SSC-A, much like proliferating cells (Fig. 1f,h). The behaviour of NPs in resting versus proliferating A549 cells was also examined with respect to cell area using microscopy (Fig. 1i and Supplementary Fig. 11). Although adherent animal cells are suboptimal in the context of this study, and exhibit a complex relationship between cell area and volume, we used this metric to demonstrate that the dependence of cellular NPs on cell area in resting and proliferating A549 cells is similar. Importantly, the amount of cellular NPs in S-phaseand prometaphase-arrested L1210 cells sharing the same SSC-A values was similar (Fig. 1g), demonstrating that the cellular NP amount is dependent on cell size, not cell-cycle position. We speculated that the rate of NP internalization is proportional to cell size and not constant, as has been suggested9. To test that, L1210 cells were labelled with DNA stain, cultured with NPs for 0 to 120 min, and analysed. Cells were found to internalize NPs at different rates that could be ranked by cell-cycle phases G2/M > S > G1; or by size category: large > medium > small (defined by SSC-A) (Fig. 1j). The dependence of NP uptake on SSC-A within each cell-cycle phase and the correlation between NP and cell volume after 40-min incubation (estimated by both SSC-A and microscopy) emphasize that in this context, the correlation with cell-cycle progression is an epiphenomenon of volume increase (Fig. 1k, Supplementary Figs 12,13). Altogether, we show that the NP amount in a cell is largely dependent on its size, regardless of whether the cell proliferates or not. In proliferating cells, the cellular NP amount can be ranked according to G2/M > S > G1; however, this is only a reflection of size increase, and, overall, it would be misleading to relate the behaviours of NPs studied here and by Kim et al.9 The interface of nanoparticles with proliferating mammalian cells

Gas2l3 is essential for brain morphogenesis and development

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. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype 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.

Unbiased transcriptome signature of in vivo cell proliferation reveals pro- and antiproliferative gene networks

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 G1–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 and human data sets.

Purifying Cytokinetic Cells from an Asynchronous Population

Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells. Like any other aspect of cell cycle research, the study of cytokinesis relies heavily on cell synchronization. However, the synchronization of cells during cytokinesis is challenging due to the rapid nature of this process and the shortage of cell cycle blocking agents specifically targeting this phase. Here, we demonstrate the use of standard flow cytometry for directly isolating cytokinetic cells from an asynchronous population of normally proliferating cells. This approach is based on a cell cycle marker whose temporal proteolysis, in combination with DNA quantification or cell size approximation, distinguishes cells undergoing cytokinesis. Furthermore, by avoiding doublet discrimination, typically used in flow cytometry analyses, we were able to further increase selectivity, specifically purifying cells at late cytokinesis. Our method circumvents checkpoint activation, cell cycle arrest, and any other means of pre-synchronization. These qualities, as demonstrated for both unattached and adherent cells, enable high selectivity for cytokinetic cells despite their overall low abundance in an asynchronous population. The sorted cells can then be readily used for cell biological, biochemical, and genomic applications to facilitate cytokinesis and cell cycle research.