Isolation of Protein Complexes Involved in Mitosis and Cytokinesis from Drosophila Cultured Cells

2009 ◽  
pp. 99-112 ◽  
Author(s):  
Pier Paolo D’Avino ◽  
Vincent Archambault ◽  
Marcin R. Przewloka ◽  
Wei Zhang ◽  
Ernest D. Laue ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Complexes

scholarly journals Analysis of Notch Lacking the Carboxyl Terminus Identified in Drosophila Embryos

2000 ◽  
Vol 149 (3) ◽  
pp. 683-696 ◽  
Author(s):  
Cedric S. Wesley ◽  
Lino Saez
Keyword(s):  
Lateral Inhibition ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Precursor Cells ◽  
Cell Surface Receptor ◽  
Carboxyl Terminus ◽  
Embryonic Cells ◽  
Surface Receptor ◽  
The Central Nervous System ◽  
Carboxy Terminal

The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate NΔCterm.

Interaction between the guanylate kinase domain of PSD-95 and the proline-rich region and microtubule binding repeats 2 and 3 of tau

2021 ◽  
Author(s):  
Emmanuel Prikas ◽  
Holly Ahel ◽  
Kristie Stefanoska ◽  
Prita Riana Asih ◽  
Alexander Volkerling ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Complexes ◽  
Kinase Domain ◽  
Rich Region ◽  
Guanylate Kinase ◽  
Synaptic Density ◽  
Microtubule Binding ◽  
Key Factor ◽  
Post Synaptic Density ◽  
Proline Rich Region

The microtubule-associated protein tau is a key factor in neurodegenerative proteinopathies and is predominantly found in the neuronal axon. However, somatodendritic localization of tau occurs for a subset of pathological and physiologic tau. Dendritic tau can localize to post-synapses where it interacts with proteins of the post-synaptic density (PSD) protein PSD-95, a membrane-associated guanylate kinase (MAGUK) scaffold factor for organization of protein complexes within the PSD, to mediate downstream signals. The sub-molecular details of this interaction, however, remain unclear. Here, we use interaction mapping in cultured cells to demonstrate that tau interacts with the guanylate kinase (GUK) domain in the C-terminal region of PSD-95. The PSD-95 GUK domain is required and sufficient for a complex with full-length human tau. Mapping the interaction of the MAGUK core on tau revealed the microtubule binding repeats 2 and 3 and the proline-rich region contribute to this interaction, while the N- and C-terminal regions of tau inhibit interaction. These results reveal intramolecular determinants of the protein complex of tau and PSD-95 and increase our understanding of tau interactions regulating neurotoxic signaling at the molecular level.

scholarly journals Caveolae protect endothelial cells from membrane rupture during increased cardiac output

2015 ◽  
Vol 211 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Jade P.X. Cheng ◽  
Carolina Mendoza-Topaz ◽  
Gillian Howard ◽  
Jessica Chadwick ◽  
Elena Shvets ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cardiac Output ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Membrane Integrity ◽  
Hemodynamic Forces ◽  
Membrane Rupture ◽  
Acute Rupture

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.

scholarly journals Activity of the mouse Notch ligand DLL1 is sensitive to C-terminal tagging in vivo

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Karin Schuster-Gossler ◽  
Karsten Boldt ◽  
Dorothee Bornhorst ◽  
Patricia Delany-Heiken ◽  
Marius Ueffing ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cho Cells ◽  
Vertebral Column ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Developmental Processes ◽  
Notch Ligand ◽  
Direct Fluorescence

Abstract Objective The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. Results We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 4777-4785 ◽  
Author(s):  
Stefan Amatschek ◽  
Ernst Kriehuber ◽  
Wolfgang Bauer ◽  
Barbel Reininger ◽  
Paul Meraner ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Lymphatic Endothelial Cell ◽  
Class Ii ◽  
Fluid Exchange ◽  
Cell Functions ◽  
Marker Proteins

Abstract The discovery of marker proteins of human blood (BECs) and lymphatic endothelial cells (LECs) has allowed researchers to isolate these cells. So far, efforts to unravel their transcriptional and functional programs made use of cultured cells only. Hence, it is unknown to which extent previously identified LEC- and BEC-specific programs are representative of the in vivo situation. Here, we define the human BEC- and LEC-specific in vivo transcriptomes by comparative genomewide expression profiling of freshly isolated cutaneous EC subsets and of non-EC skin cells (fibroblasts, mast cells, dendritic cells, epithelial cells). Interestingly, the expression of most of the newly identified EC subset-discriminating genes depends strictly on the in vivo tissue environment as revealed by comparative analyses of freshly isolated and cultured EC subsets. The identified environment-dependent, EC subset-restricted gene expression regulates lineage fidelity, fluid exchange, and MHC class II–dependent antigen presentation. As an example for a BEC-restricted in vivo function, we show that non-activated BECs in situ, but not in vitro, assemble and display MHC class II protein complexes loaded with self-peptides. Thus, our data demonstrate the key importance of using precisely defined native ECs for the global identification of in vivo relevant cell functions.

scholarly journals Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development

2019 ◽  
Vol 218 (10) ◽  
pp. 3455-3471 ◽  
Author(s):  
Jingyi Gong ◽  
Thomas N. Gaitanos ◽  
Olivia Luu ◽  
Yunyun Huang ◽  
Louise Gaitanos ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Protein Clusters ◽  
Nucleotide Exchange Factor

Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1’s presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.

scholarly journals Endocytic Clathrin Coats Develop Curvature at Early Stages of Their Formation

2019 ◽  
Author(s):  
Nathan M. Willy ◽  
Joshua P. Ferguson ◽  
Salih Silahli ◽  
Cemal Cakez ◽  
Farah Hasan ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Protein Complexes ◽  
Membrane Curvature ◽  
Coated Vesicle ◽  
Endocytic Vesicle ◽  
Coated Pits ◽  
Early Stages ◽  
Pit Formation ◽  
Endocytic Protein ◽  
Different Levels

AbstractSculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of endocytic protein complexes. The mechanism through which membrane curvature is imposed during formation of clathrin-coated vesicles is an ongoing controversy. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by clathrin-coated pits can be detected in real time within cultured cells and tissues of developing metazoan organisms. We found that the footprint of clathrin coats increase monotonically during formation of curved pits at different levels of plasma membrane tension. Our findings are only compatible with models that predict curvature generation at early stages of endocytic clathrin-coated pit formation. Therefore, clathrin-coated vesicle formation does not necessitate a dynamically unstable clathrin lattice that would allow an abrupt flat-to-curved transition.SummaryEndocytic clathrin coats acquire curvature without a flat-to-curved transition that requires an extensive reorganization of the clathrin lattice.

Inheritance of phenotype in mammalian cells: genetic vs. epigenetic mechanisms

1991 ◽  
Vol 260 (6) ◽  
pp. L386-L394 ◽  
Author(s):  
D. C. Gruenert ◽  
A. L. Cozens
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Growth Regulation ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Epigenetic Mechanisms ◽  
Phenotypic Changes ◽  
Pathways Analysis

Inherited phenotypic changes in cultured cells, as observed during differentiation and transformation, reflect alterations in gene expression and have both a genetic and epigenetic basis. The causes of specific changes are often difficult to define especially when observing phenomenological end points. Although such observations are an important step in defining the phenotypic changes that endure for multiple generations, it is necessary to analyze cells at the molecular level to characterize the pathways leading to changes in phenotype. Gene expression can be regulated at multiple levels, i.e., DNA structure, gene transcription, and/or posttranscriptional modifications. Four genetic mechanisms (DNA point mutations, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation and the preservation of DNA-protein complexes) can account for the majority of enduring changes observed in cultured cells. Genetic alterations in DNA sequence appear to be largely responsible for altered growth regulation associated with transformation, but there is also evidence to suggest that epigenetic mechanisms play a role in transformation. Differentiation of cultured cells is often associated with lack of growth, and has been ascribed in part to epigenetic mechanisms. However, differentiation and transformation are not mutually exclusive but may be regulated by parallel multistep pathways. Analysis of the causes underlying transformation has been complicated by the use of aneuploid cells, but it is clear that the tools for overcoming the ambiguities associated with phenomenological analysis are available.

scholarly journals The C-Terminal Tail of Yersinia pseudotuberculosis YopM Is Critical for Interacting with RSK1 and for Virulence

2010 ◽  
Vol 78 (6) ◽  
pp. 2584-2598 ◽  
Author(s):  
Melissa W. McCoy ◽  
Meghan L. Marré ◽  
Cammie F. Lesser ◽  
Joan Mecsas
Keyword(s):  
Nuclear Localization ◽  
Yersinia Pseudotuberculosis ◽  
Immune Cell ◽  
Cultured Cells ◽  
Nk Cell ◽  
Protein Complexes ◽  
Host Cells ◽  
Cell Populations ◽  
Independent Functions ◽  
Ribosomal S6 Kinase

ABSTRACTYersiniaspp. undermine the immune responses of infected animals by translocating Yops directly into host cells with a type III secretion system. YopM, a leucine-rich repeat protein, is a critical virulence factor in infection. YopM localizes to both the nucleus and the cytoplasm in cultured cells, interacts with mammalian p90 ribosomal S6 kinase 1 (RSK1), and causes a decrease in NK cell populations in spleens. Little is known about the molecular interaction between YopM and RSK1 and its significance in pathogenesis. We performed a systematic deletion analysis of YopM inYersinia pseudotuberculosisto determine which regions are required for RSK1 interactions, nuclear localization, virulence, and changes in immune cell populations during infection of mice. Full-length YopM associated with RSK1 in at least two protein complexes in infected cells, and deletion of its C-terminal tail abrogated all RSK1 interactions. The C-terminal tail was required for tissue colonization, asyopMmutants that failed to interact with RSK1 were as defective for tissue colonization as was a ΔyopMmutant; however, nuclear localization of YopM was not dependent on its RSK1 interaction. Mutants expressing YopM proteins which do not interact with RSK1 caused more pathology than did the ΔyopMmutant, suggesting that there are other RSK1-independent functions of YopM. Histopathological and flow cytometric analyses of spleens showed that infection with wild-typeY. pseudotuberculosiscaused an influx of neutrophils, while mice infected withyopMmutants had increased numbers of macrophages. Decreases in NK cells afterY. pseudotuberculosisinfection did not correlate with YopM expression. In conclusion, the C terminus of YopM is essential for RSK1 interactions and for virulence.

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