scholarly journals A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation

2020 ◽  
Vol 6 (16) ◽  
pp. eaaz9899
Author(s):  
Yong Chi ◽  
John H. Carter ◽  
Jherek Swanger ◽  
Alexander V. Mazin ◽  
Robert L. Moritz ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Complexes ◽  
Nuclear Architecture ◽  
Cyclin Dependent Kinase ◽  
Oncogenic Signaling ◽  
Cellular Processes ◽  
Validation Rate ◽  
Associated Proteins ◽  
Epigenetic Regulators

Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an “in situ” approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5′-triphosphate analogs. We identified 117 candidate substrates, ~40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.

scholarly journals ZapA tetramerization is required for midcell localization and ZapB interaction in Escherichia coli

2019 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Tanneke den Blaauwen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacterial Cell Division ◽  
Cellular Processes ◽  
Interaction Partners ◽  
Associated Proteins

AbstractBacterial cell division is guided by FtsZ treadmilling precisely at midcell. FtsZ itself is regulated by FtsZ associated proteins (Zaps) that couple it to different cellular processes. ZapA is known to enhance FtsZ bundling but also forms the synchronizing link with chromosome segregation through ZapB and matS bound MatP. ZapA exists as dimers and tetramers in the cell. Using the ZapAI83E mutant that only forms dimers, this paper investigates the effects of ZapA multimerization state on its interaction partners and cell division. By employing (fluorescence) microscopy and Förster Resonance Energy Transfer in vivo it is shown that; dimeric ZapA is unable to complement a zapA deletion strain and localizes diffusely through the cell but still interacts with FtsZ that is not part of the cell division machinery. Dimeric ZapA is unable to recruit ZapB, which localizes in its presence unipolarly in the cell. Interestingly, the localization profiles of the chromosome and unipolar ZapB anticorrelate. The work presented here confirms previously reported in vitro effects of ZapA multimerization in vivo and further places it in a broader context by revealing the strong implications for ZapB localization and ter linkage.

scholarly journals Mapping Cell Membrane Organization and Dynamics Using Soft Nano-Imprint Lithography

2019 ◽  
Author(s):  
T. Sansen ◽  
D. Sanchez-Fuentes ◽  
R. Rathar ◽  
A. Colom-Diego ◽  
F. El Alaoui ◽  
...  
Keyword(s):  
Cell Membrane ◽  
High Performance ◽  
Protein Complexes ◽  
Membrane Mechanics ◽  
Nanostructured Surfaces ◽  
Cellular Processes ◽  
State Organization ◽  
Cell Membrane Mechanics ◽  
Membrane Shape

AbstractMembrane shape is a key feature of many cellular processes, including cell differentiation, division, migration, and trafficking. The development of nanostructured surfaces allowing for the in situ manipulation of membranes in living cells is crucial to understand these processes, but this requires complicated and limited-access technologies. Here, we investigate the self-organization of cellular membranes by using a customizable and bench top method allowing to engineer 1D SiO2 nanopillar arrays of defined sizes and shapes on high-performance glass compatible with advanced microscopies. As a result of this original combination, we provide a mapping of the morphology-induced modulation of the cell membrane mechanics, dynamics and steady-state organization of key protein complexes implicated in cellular trafficking and signal transduction.

scholarly journals Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

2020 ◽  
Vol 21 (7) ◽  
pp. 2283 ◽  
Author(s):  
Katarzyna Kwiatkowska ◽  
Orest V. Matveichuk ◽  
Jan Fronk ◽  
Anna Ciesielska
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Hydrophobic Interactions ◽  
Protein S ◽  
Oncogenic Signaling ◽  
Cellular Processes ◽  
Sphingosine 1 Phosphate ◽  
Recent Developments ◽  
Associated Proteins ◽  
Mutual Relations

Flotillin-1 and flotillin-2 are ubiquitously expressed, membrane-associated proteins involved in multifarious cellular events from cell signaling, endocytosis, and protein trafficking to gene expression. They also contribute to oncogenic signaling. Flotillins bind the cytosolic leaflet of the plasma membrane and endomembranes and, upon hetero-oligomerization, serve as scaffolds facilitating the assembly of multiprotein complexes at the membrane–cytosol interface. Additional functions unique to flotillin-1 have been discovered recently. The membrane-binding of flotillins is regulated by S-palmitoylation and N-myristoylation, hydrophobic interactions involving specific regions of the polypeptide chain and, to some extent, also by their oligomerization. All these factors endow flotillins with an ability to associate with the sphingolipid/cholesterol-rich plasma membrane domains called rafts. In this review, we focus on the critical input of lipids to the regulation of the flotillin association with rafts and thereby to their functioning. In particular, we discuss how the recent developments in the field of protein S-palmitoylation have contributed to the understanding of flotillin1/2-mediated processes, including endocytosis, and of those dependent exclusively on flotillin-1. We also emphasize that flotillins affect directly or indirectly the cellular levels of lipids involved in diverse signaling cascades, including sphingosine-1-phosphate and PI(4,5)P2. The mutual relations between flotillins and distinct lipids are key to the regulation of their involvement in numerous cellular processes.

scholarly journals Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Chia-Yu Guh ◽  
Yu-Hung Hsieh ◽  
Hsueh-Ping Chu
Keyword(s):  
Protein Complexes ◽  
Nuclear Organization ◽  
Regulation Of Gene Expression ◽  
Nuclear Architecture ◽  
Living Cells ◽  
Cellular Processes ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Non Coding Rnas

AbstractProtein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo.

scholarly journals CyLaKS: the Cytoskeleton Lattice-based Kinetic Simulator

2021 ◽  
Author(s):  
Shane A. Fiorenza ◽  
Meredith D. Betterton ◽  
Daniel G. Steckhahn
Keyword(s):  
Cell Division ◽  
Detailed Balance ◽  
Cell Movement ◽  
Spatiotemporal Variation ◽  
Binding Kinetics ◽  
Self Organization ◽  
Simulation Framework ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Cytoskeletal Networks

Interaction of cytoskeletal filaments, motor proteins, and crosslinkers drives important cellular processes including cell division and cell movement. Cytoskeletal networks also undergo nonequilibrium self-organization in reconstituted systems. An emerging problem in cytoskeletal modeling and simulation is spatiotemporal alteration of the dynamics of filaments, motors, and associated proteins. This can occur due to motor crowding and obstacles along filaments, motor interactions and direction switching, and changes, defects, and heterogeneity in the filament lattice. How such spatiotemporally varying cytoskeletal filaments and motor interactions affect their collective properties is not fully understood. We developed the Cytoskeleton Lattice-based Kinetic Simulator (CyLaKS) for problems with significant spatiotemporal variation of motor or filament properties. The simulation builds on previous work modeling motor mechanochemistry into a simulation with many interacting motors and/or associated proteins. CyLaKS also includes detailed-balance in binding kinetics and movement and lattice heterogeneity. The simulation framework is flexible and extensible for future modeling work. Here we illustrate use of CyLaKS to study long-range motor interactions, filament heterogeneity, motion of a heterodimeric motor, and how changing crosslinker number affects filament separation.

scholarly journals Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

2019 ◽  
Vol 20 (14) ◽  
pp. 3376 ◽  
Author(s):  
Kimberly Cramer ◽  
Anna-Lena Bolender ◽  
Iris Stockmar ◽  
Ralf Jungmann ◽  
Robert Kasper ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Bacterial Cells ◽  
Sted Microscopy ◽  
E Coli ◽  
Cellular Processes

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

scholarly journals Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2718
Author(s):  
María González-González ◽  
José María Sayagués ◽  
Luis Muñoz-Bellvís ◽  
Carlos Eduardo Pedreira ◽  
Marcello L. R. de Campos ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Utility ◽  
Genetic Alterations ◽  
Western World ◽  
Sporadic Colorectal Cancer ◽  
Protein Arrays ◽  
Humoral Immune ◽  
Cellular Processes ◽  
Healthy Donors ◽  
Associated Proteins

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

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