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  • 1
    Keywords: SACCHAROMYCES-CEREVISIAE ; CELL-CYCLE ; YEAST ; PLASMA-MEMBRANE ; DEGRADATION ; NUCLEAR-PORE COMPLEX ; Half-Life ; UBIQUITIN LIGASES ; MONOMERIC RED ; END RULE PATHWAY
    Abstract: The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule-mediated protein degradation.Systematic monitoring of proteome dynamics would require simultaneous measurement of protein turnover and subcellular trafficking at the single-cell and population scales. The importance of protein turnover was introduced in 1942 by Schonheimer, who noted that "all constituents of living matter, whether functional or structural, of simple or of complex constitution, are in a steady state of rapid flux". Protein homeostasis is now understood as a balance between protein synthesis, through transcription and translation, and protein degradation, through processes such as proteasomal and lysosomal degradation, tuned in response to intrinsic and extrinsic inputs. Alterations in protein turnover are observed in aging organisms and underlie various diseases. Deregulated degradation of cell cycle control proteins such as the p53 tumor suppressor plays a critical role in many forms of human cancers. Abnormal trafficking and degradation of a mutant form of a chloride ion channel causes cystic fibrosis. Moreover, accumulation of specific proteins is linked to neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. Therefore, understanding protein turnover and mobility could provide new strategies for targeted clinical interference to treat such diseases.
    Type of Publication: Journal article published
    PubMed ID: 22729030
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  • 2
    Keywords: SACCHAROMYCES-CEREVISIAE ; BUDDING YEAST ; PLASMA-MEMBRANE ; PROTEIN INTERACTIONS ; fluorescence correlation spectroscopy ; NEGATIVE REGULATION ; ARP2/3 COMPLEX ; COATED PITS ; CLATHRIN-MEDIATED ENDOCYTOSIS ; CORTICAL ACTIN CYTOSKELETON
    Abstract: Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.
    Type of Publication: Journal article published
    PubMed ID: 25366307
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  • 3
    Keywords: SACCHAROMYCES-CEREVISIAE ; CELL-CYCLE ; CRYSTAL-STRUCTURE ; FISSION YEAST ; SCHIZOSACCHAROMYCES-POMBE ; CHROMOSOME INSTABILITY ; ANAPHASE-PROMOTING COMPLEX ; MITOTIC CHECKPOINT ; CYCLE-REGULATED GENES ; BIOLOGICAL NOISE
    Abstract: The spindle assembly checkpoint is a conserved signalling pathway that protects genome integrity. Given its central importance, this checkpoint should withstand stochastic fluctuations and environmental perturbations, but the extent of and mechanisms underlying its robustness remain unknown. We probed spindle assembly checkpoint signalling by modulating checkpoint protein abundance and nutrient conditions in fission yeast. For core checkpoint proteins, a mere 20% reduction can suffice to impair signalling, revealing a surprising fragility. Quantification of protein abundance in single cells showed little variability (noise) of critical proteins, explaining why the checkpoint normally functions reliably. Checkpoint-mediated stoichiometric inhibition of the anaphase activator Cdc20 (Slp1 in Schizosaccharomyces pombe) can account for the tolerance towards small fluctuations in protein abundance and explains our observation that some perturbations lead to non-genetic variation in the checkpoint response. Our work highlights low gene expression noise as an important determinant of reliable checkpoint signalling.
    Type of Publication: Journal article published
    PubMed ID: 24161933
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  • 4
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    German Medical Science; Düsseldorf, Köln
    In:  Evidenzbasierte Medizin - Anspruch und Wirklichkeit; 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft; 20040923-20040926; Berlin; DOC04dogDO.01.03 /20040922/
    Publication Date: 2004-09-21
    Keywords: ddc: 610
    Language: English
    Type: conferenceObject
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