Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes

Challenges and Opportunities for Ecosystem-Based Management and Marine Spatial Planning in the Irish Sea

Ecosystem-Based Management (EBM) integrates the connections between land, air, water and all living things including human beings and their institutions. The location of the Irish Sea, between major historical industrial centres, its history of use and exploitation, combined with its hydrographic characteristics, have led to the current patterns of use. EBM efforts have been ongoing for over a decade but political boundaries have led to fragmented governance. The forthcoming UK exit from the European Union (EU) may pose further challenges. This chapter examines articulations between political boundaries, spatial scales of Marine Spatial Planning and nested social-ecological systems including the gyre in the western Irish Sea, and Dublin Bay. Examples of emerging best practices are provided and the challenges of data availability for ecosystem services are considered

Cell cycle-regulated protein abundance changes in synchronously proliferating HeLa cells include regulation of pre-mRNA splicing proteins.

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes

Top three significant GO terms enriched in the individual lists of MG132-sensitive proteins.

<p>Top three significant GO terms enriched in the individual lists of MG132-sensitive proteins.</p

Discordance between mRNA and protein abundance.

<p>A) Individual lists of proteins that changed by at least 1.5-fold were compared to the mRNA data for those same proteins in synchronized HeLa cells from Whitfield et al. 2002 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Whitfield1" target="_blank">[7]</a>. The percentage of proteins whose corresponding mRNA also changed is graphed for both S phase and G2 phase. ** p<0.001. B-E) Individual lists of proteins that changed by at least 1.5-fold were compared to proteins predicted to be B) ubiquitinated in asynchronous HCT116 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Kim1" target="_blank">[15]</a>, C) phosphorylated in HeLa cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Olsen1" target="_blank">[8]</a>, D) substrates of Cyclin A/Cdk2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Chi1" target="_blank">[17]</a>, and E) substrates of the ATR kinase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Stokes1" target="_blank">[16]</a>. The percentage of each list that overlaps with the published dataset is plotted. * p<0.01; ** p<0.001.</p

Top three significant GO terms enriched in individual lists of cell cycle-regulated proteins.

<p>Top three significant GO terms enriched in individual lists of cell cycle-regulated proteins.</p

Validation of selected cell cycle-regulated protein predicted by mass spectrometry.

<p>The same cell lysates analyzed by mass spectrometry were subjected to immunoblot analysis for the indicated endogenous proteins in the A) G1 to S lysates or B) S to G2 lysates. Reported fold change ratios from mass spectrometry are listed to the right.</p

Cell cycle-regulated proteins from G1 to S and S to G2 detected by mass spectrometry.

<p>A) Comparison of the total number of proteins detected in this study (2,842 proteins) to two other studies of the HeLa cell proteome: Nagaraj et al., 2011 (10,237 proteins) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Nagaraj1" target="_blank">[39]</a> and Olsen et al., 2010 (6,695 proteins) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058456#pone.0058456-Olsen1" target="_blank">[8]</a>. B) Quantified proteins from this study were divided into lists based on their fold and direction of change; the total protein count for each list is plotted. “NC” denotes proteins that did not change. “NC MG,” “Inc MG,” and “Dec MG” denote proteins that either did not change, increased, or decreased in response to MG132 treatment, respectively. C) All quantifiable proteins in the G1 to S dataset plotted by their log₂ transformed isotope ratios (medium S phase/light G1 phase). Dotted lines denote the 1.5-fold change threshold. D) All quantifiable proteins identified in the S to G2 dataset plotted by their log₂ transformed isotope ratios (medium G2 phase/light S phase); dotted lines denote the 1.5-fold change threshold. E) Proteins identified in early-S phase cells compared to early-S phase cells treated with MG132 plotted by their log₂ transformed isotope ratios (heavy S phase plus MG132/medium S phase minus MG132). Dotted lines denote the 1.5-fold change threshold. F) Proteins identified in G2 phase cells compared to G2 phase cells treated with MG132 plotted by their log₂ transformed isotope ratios (heavy G2 plus MG132/medium G2 phase minus MG132). Dotted lines denote the 1.5-fold change threshold.</p

HeLa cell synchronization.

<p>A) Cells were synchronized by a modified double-thymidine block then released by re-plating and harvested at the indicated time points; a late G1 phase culture was treated with MG132 two hrs prior to harvest in early S phase. Synchrony was determined by flow cytometric analysis of DNA content. B) Immunoblot analysis of endogenous Cyclin A, Cdt1, SLBP, and tubulin proteins in whole cell lysates from portions of the same cells used in A. C) Cells were metabolically labeled with stable isotopes and then synchronized in G1 (3 hrs after mitosis, normal/“light” isotopes) and early-S phase (10 hrs after mitosis, labeled with intermediate or “medium” isotopes) as in A and B. Cells labeled with the heaviest isotopes were treated with MG132 two hrs prior to harvest in early S phase. Immunoblot analysis of endogenous Cdc6, Cdt1, and geminin in whole cell lysates used for subsequent mass spectrometric tests. A non-specific band (NSB) serves as a loading control. D) Cells were synchronized by double-thymidine block, released into S phase, and harvested at the indicated timepoints. Synchrony was determined by flow cytometric analysis of DNA content. E) Immunoblot analysis of endogenous Cyclin B, SLBP and Cdt1 in whole cell lysates from portions of the same cells used in D. F) Cells were metabolically labeled with stable isotopes and synchronized in S phase (light isotopes) or G2 phase (medium isotopes) as in D and E. A culture labeled with heavy isotopes was treated with MG132 in late S phase for two hrs prior to harvest in G2. Immunoblot analysis of endogenous Cdt1 and SLBP in whole cell lysates used for subsequent mass spectrometric analysis; β-actin serves as a loading control.</p