past and present
+ tandem fluorescent timer proteins (tFTs)
"measure protein age and protein turnover in space and time"
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.
Khmelinskii A, Keller PJ, Bartosik A, Meurer M, Barry JD, Mardin BR, Kaufmann A, Trautmann S, Wachsmuth M, Pereira G, Huber W, Schiebel E & Knop M (2012) Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat. Biotechnol. 30: 708–714
+ Protein quality control in the nucleus
Removal of mis-targeted proteins from the inner nuclear membrane
We identified a protein degradation pathway at the INM in yeast mediated by the Asi complex. The Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.
Khmelinskii A, Blaszczak E, Pantazopoulou M, Fischer B, Omnus DJ, Le Dez G, Brossard A, Gunnarsson A, Barry JD, Meurer M, Kirrmaier D, Boone C, Huber W, Rabut G, Ljungdahl PO & Knop M (2014) Protein quality control at the inner nuclear membrane. Nature 516: 410–413
+ N-end rule pathway
Systematic peptide profiling reveales the logic of N-degrons
In this study we systematically examined how protein turnover is affected by N-terminal sequences. We performed a comprehensive survey of degrons in the yeast N-terminome using a high-throughput degron profiling approach based on the tandem fluorescent protein timer (tFT). We found that approximately 26% of nascent protein N termini encode cryptic de- grons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identified two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1.
Kats I, Khmelinskii A, Kschonsak M, Huber F, Knieß RA, Bartosik A & Knop M (2018) Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome. Mol. Cell 70: 488–501.e5
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+ Libraries for high throughput yeast cell biology
Arrayed libraries: SWAT
We constructed an arrayed C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF. This module can be efficiently replaced with tags or regulatory elements. We tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
Meurer M, Duan Y, Sass E, Kats I, Herbst K, Buchmuller BC, Dederer V, Huber F, Kirrmaier D, Stefl M, Van Laer K, Dick TP, Lemberg MK, Khmelinskii A, Levy ED & Knop M (2018) Genome-wide C-SWAT library for high-throughput yeast genome tagging. Nat Methods 15: 598–600
Pooled libraries: CASTLING
Construction of arrayed libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we developed CRISPR/Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones.
Buchmuller BC, Herbst K, Meurer M, Kirrmaier D, Sass E, Levy ED & Knop M (2018) Pooled clone collections by multiplexed CRISPR/Cas12a-assisted gene tagging in yeast. bioRxiv: 476804, doi: https://doi.org/10.1101/476804
+ Light sheet microscopes
live cell imaging without bleaching (almost)
In this publication we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements. We use it to visualise the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue.
Capoulade J, Wachsmuth M, Hufnagel L & Knop M (2011) Quantitative fluorescence imaging of protein diffusion and interaction in living cells. Nat. Biotechnol. 29: 835–839
The πSPIM microscope
In this work we demonstrate the use of an oblique light-sheet configuration adapted to provide the highest possible Gaussian beam enabled resolution in light sheet fluorescence microscopy (LSFM).
The oblique light-sheet configuration furthermore enables LSFM imaging at the surface of a cover slip, without the need of specific sample mounting. In addition, the system is compatible with simultaneous high NA wide-field epi-fluorescence imaging of the specimen contained in a glass-bottom cell culture dish.
Theer P, Dragneva D & Knop M (2016) πSPIM: high NA high resolution isotropic light-sheet imaging in cell culture dishes. Scientific Reports 6: 32880
The light pad microscope