The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed ‘early risers’. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

(150/150 words) 21The temporal regulation of protein abundance and post-translational modifications is a 22 key feature of cell division. Recently, we analysed gene expression and protein 23 abundance changes during interphase under minimally perturbed conditions (Ly et al. 24 2014;Ly et al. 2015). Here we show that by using specific intracellular immunolabeling 25 protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, 26 can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS 27 (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now 28 compare protein abundance and phosphorylation changes in interphase and mitotic 29 fractions from asynchronously growing human cells. We identify a set of 115 30 phosphorylation sites increased during G2, which we term 'early risers'. This set 31 includes phosphorylation of S738 on TPX2, which we show is important for TPX2 32 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-33 wide analysis of protein abundance remodeling between prophase, prometaphase and 34 anaphase. 35 36. CC-BY 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/125831 doi: bioRxiv preprint first posted online Apr. 9, 2017; 3 Introduction 37 38The mitotic cell division cycle is composed of four major phases, i.e., G1, S, G2 39 and M. The phases are defined by two major events during cell division: DNA replication 40 (S phase) and mitosis (M phase), with intervening gap phases (G1 and G2). The cell 41 cycle is driven by the expression of key proteins, called cyclins. Generally, cyclin 42 expression and function is restricted to specific cell cycle phases, driving temporally 43 ordered phosphorylation of key substrates by interacting with their kinase partners, the 44 cyclin dependent kinases (CDKs). Temporally regulated degradation of the cyclins 45 ensures that progression through the cell cycle is unidirectional. For example, cyclin A 46 expression increases during S-phase, reaching a maximum in mitosis. During 47 prometaphase, cyclin A is targeted for degradation by the anaphase promoting 48 complex/cyclosome (APC/C), a multiprotein E3 ubiquitin ligase, thus restricting cyclin 49 A-driven phosphorylation to S, G2 and early M-phase. 50Mitosis can be further resolved into subphases (i.e. prophase, prometaphase, 51 metaphase, anaphase, telophase & cytokinesis), which are characterised by the 52 widespread reorganization of subcellular architecture. For example, in prophase, 53 duplicated centrosomes separate to form the poles of the mitotic spindle. Centrosome 54 separation is dependent on the activities of several kinases, including Cdk1 and Plk1 55 (Smith et al. 2011), and on the microtubule motor protein Eg5 (Sawin et al. 1992). 56Improperly timed centrosome separation can lead to chrosomomal instability, as shown 57 in cyclin B2-overexpressing MEFs (Nam & v...

Using high resolution quantitative mass spectrometry we have generated in-depth proteome maps of human and mouse ex vivo and in vitro microglia. We reveal a tenfold difference in protein content of ex vivo and in vitro cells and fundamental differences in protein expression related to protein synthesis, metabolism, microglia markers and environmental sensors. While human and mouse microglia are different in their proteomes, the species differences are smaller than the changes induced by cell culture. Remarkably, xenografting human microglia derived from human stem cells into mouse brain restores the in vivo-like microglia proteomic signature including reduction in the total amount of protein, energy demand and protein synthesis, and re-expression of homeostatic microglia markers. We identify more than 9000 microglia proteins and discuss how they relate to microglia function. This data provides a vital proteomic resource for understanding in vitro and ex vivo microglia phenotypes.

Immune activated T lymphocytes modulate the activity of key metabolic pathways to support the transcriptional reprograming and reshaping of cell proteomes that permits effector T cell differentiation. The present study uses high resolution mass spectrometry and metabolic labelling to explore how T cells control the methionine cycle to produce methyl donors for protein and nucleotide methylations. We show that antigen receptor engagement controls flux through the methionine cycle and also controls RNA and histone methylations. We establish that the main rate limiting step for the methionine cycle is control of methionine transporter expression by antigen receptors. Only T cells that respond to antigen to upregulate and sustain methionine transport are supplied with the methyl donors that permit the dynamic nucleotide methylations and epigenetic reprogramming that drives T cell differentiation.

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014(Ly et al., , 2015. Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.