Single Live Cell Monitoring of Protein Turnover Reveals Intercellular Variability and Cell-Cycle Dependence of Degradation Rates

Cells need to reliably control their proteome composition to maintain homeostasis and regulate growth. How protein synthesis and degradation interplay to control protein expression levels remains unclear. Here, we combined a tandem fluorescent timer and pulse-chase protein labeling to disentangle how protein synthesis and degradation control protein homeostasis in single live mouse embryonic stem cells. We discovered substantial cell-cycle dependence in protein synthesis rates and stabilization of a large number of proteins around cytokinesis. Protein degradation rates were highly variable between cells, co-varied within individual cells for different proteins, and were positively correlated with synthesis rates. This suggests variability in proteasome activity as an important source of global extrinsic noise in gene expression. Our approach paves the way toward understanding the complex interplay of synthesis and degradation processes in determining protein levels of individual mammalian cells.

Single live cell monitoring of protein turnover reveals intercellular variability and cell cycle dependence of degradation rates

Andrea Brigitta Alber

Cells need to be able to precisely control their proteome composition in order to maintain homeostasis. This is of particular relevance in fast dividing cells, such as embryonic stem cells, since their global protein levels need to be doubled at each division to ensure reliable growth and pluripotency maintenance. However, how protein synthesis and degradation interplay to regulate global protein expression levels throughout the cell cycle remains unclear. In addition, while cell-to-cell variability at the level of transcription and protein expression is relatively well studied, it remains unknown to what extent protein degradation rates vary between single cells. Previous studies focusing on protein turnover have mainly relied on bulk biochemical approaches, thus only providing population averages and a low temporal resolution. In addition, many approaches to study the changes in protein turnover throughout the cell cycle have been dependent on cell cycle synchonizing drugs, which might interfere with protein synthesis and also reduce cell viability. Therefore, we further developed and applied two fluorescence time-lapse imaging strategies to study the cell cycle dependence and cell-to-cell variability of protein synthesis and degradation in unperturbed dividing mouse embryonic stem (ES) cells: 1) a Mammalian Cell-optimized Tandem Fluorescent Timer (MCFT), consisting of the fusion of the fast maturing green fluorescent protein superfolder GFP (sfGFP) and the slower maturing red fluorescent protein mOrange2, which in combination with computational modeling allows for disentangling changes in protein synthesis from changes in protein degradation in single living cells, and 2) fluorescent pulse labeling of protein tags (SNAP/Halo-tags) to monitor protein half-lives, as well as transient changes in protein degradation rates in single cells without the use of protein synthesis inhibitors. We discovered a substantial cell cycle-dependence in both protein synthesis and degradation. In particular, a large fraction (40%) of proteins was stabilized around cytokinesis, an effect that has not been described previously. Interestingly, protein degradation rates were highly variable between single cells and were positively correlated with synthesis rates in individual cells to buffer protein expression level variability. Furthermore, degradation rates of different proteins co-varied between single cells, both in ES cells and fibroblasts, suggesting variability in proteasome activity as a source of global extrinsic noise in gene expression. We identified the proteasome component ADRM1 as a potential limiting factor that contributes to generating such noise, as its expression levels were negatively correlated with protein half-lives in single cells. Our work demonstrates the importance of single cell live imaging approaches to study dynamic processes and cell-to-cell variability and sheds more light on the complex interplay of protein synthesis and degradation in determining protein expression levels in single mammalian cells.

EPFL2018

Single-Cell Analysis of Mycobacterial Persistence Using Microfabricated Tools

Meltem Elitas

Bacterial cells behave as individuals despite being genetically identical and subject to the same environment. Although the underlying mechanisms of cellular individuality are not well understood, temporal variation of gene expression and protein levels in individual cells could act as sources for phenotypic heterogeneity of populations. Bacterial persistence in fluctuating environments relies on such diversity, allowing some individuals, by chance, to survive a challenge that would kill an unadapted and phenotypically homogeneous population. Bacterial persistence is a non-genetic, metastable phenomenon that is readily lost upon subculture of surviving cells in the absence of the environmental stress. A medically relevant example of persistence is the fractional killing of bacteria by antibiotics, whereby a subpopulation of cells survives indefinitely despite prolonged exposure to a bactericidal drug. It is hypothesized in the case of tuberculosis (TB) that drug therapy may require administration of multiple drugs for 6-9 months in order to eliminate a subpopulation of "persister" cells. The focus of this thesis is to understand how persisters tolerate antibiotics that kill their genetically identical siblings. To address this question, automated time-lapse fluorescence microcopy was applied in conjunction with microfluidic and microelectromechanical systems (MEMS) to study bacterial responses to antibiotics in real time at the single-cell level. A genetic approach was employed to identify mutants of Mycobacterium smegmatis with altered rates of persistence against the first-line anti-TB drug isoniazid (INH). A microfluidic platform was designed for high-throughput screening of transposon (Tn) mutant libraries. A pilot screen of 1,100 Tn mutants revealed 11 mutants that showed increased (persister-up, PU) or decreased (persister-down, PD) survival when exposed to isoniazid. These mutants were further characterized in comparison to wild-type cells using the single-cell microfluidics platform. This single-cell analysis revealed distinct modes of persistence within each category, which were otherwise masked at the population level. The results indicate that persistence behavior was the cumulative result of the dynamics of cell division and cell lysis. Mutants with decreased persistence exhibited behavior at single-cell level that challenges the accepted mechanism of isoniazid-mediated killing. Therefore, these results yield new insights into antibiotic persistence and isoniazid's mode of action. Persisters cannot be easily studied in non-fractionated populations of cells where they represent a small percentage of the total. As a complementary approach, a method was developed based on dielectrophoretic (DEP) purification of naturally occurred low frequency persister cells from an antibiotic-treated bacterial culture. Purification of persister cells would allow for downstream analysis using conventional bulk/population level transcriptomic and proteomic approaches. The crossover frequencies for live and dead cells were determined to enable selective trapping or release from the device. This approach enabled a proof-of-concept enrichment of live bacteria in the mixture of live and dead cells after INH treatment, based on their dielectric properties. Quantitative confirmation of the enriched/isolated cell populations was attempted using flow cytometry. The high-throughput microfluidic screening and single-cell approaches developed in this thesis could, in principal, be applied to the genetic analysis of a wide variety of dynamic cellular processes that can be imaged by time- lapse fluorescence microscopy, such as gene expression, cell division, chromosome replication and segregation, and protein localization. The biochemical analysis of live cells isolated by dielectrophoresis-based separation would provide useful information about the underlying persistence mechanism. Dielectrophoresis could also be a valuable analytical tool for mutant characterization based on their intrinsic dielectric properties, either at single-cell or population levels. In conclusion, this thesis presents several new microfabricated tools to identify and understand bacterial persistence. The thesis illustrates how integration of microfluidic tools with automated time-lapse microscopy allows hidden characteristics that are present but invisible in the macro- world of batch cultures to be examined in a quantitative and high-throughput manner in the micro-world of single-cell analysis.

EPFL2012

Functional Characterization of the Mouse Olfactory Receptor mOR256-17 in Live Cells

Thamani Dahoun

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system to recognize and discriminate a nearly unlimited number of structurally diverse odorant molecules. The molecular basis of OR-mediated odor signal detection and transduction is poorly understood. This is due in part to difficulties in functional expression of ORs at high yield, preventing structural and biophysical studies at the level of the receptor protein. Here we report on the heterologous expression of the mouse receptor mOR256-17 yielding an average of 106 ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression we employed different fluorescent tags. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and posttranslational fluorescence labeling of a 12 amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. The establishment of a mOR256-17-inducible HEK293S-TetO cell line allowed us to determine by fluorescence spectrofluorometry and fluorescence correlation spectroscopy (FCS) the optimal conditions to solubilize the receptor. By screening a large odorant compound library we discovered a new selective spectrum of potent mOR256-17 specific agonists. Furthermore, by using a reporter assay that couples both Gs and Gq proteins after inhibition of adenylate cyclase by the chemical adenylyl cyclase inhibitors 9-(tetrahydro-2-furanyl)-9H-purin-6amine (SQ22536) and after the inhibition of phospholipase C by the chemical inhibitor 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), we identified one agonist which induced the inositol 1,4,5-trisphosphate (IP3) transduction pathway. We provided further evidence of the involvement of this second messenger (i) by employing another reporter gene specific to the Gq pathway and (ii) by using electrical impedance spectroscopy. Our attempt to create a FRET fluorophore pair system based on fluorescein arsenical hairpin (FlAsH) labeling and on carrier protein tags to study the receptor conformational changes during activation revealed that modifications in mOR256-17 sequences perturbs the correct receptor folding and prohibits its expression at the plasma membrane. Our study indicated that an essential prerequisite for achieving cell surface expression of mOR256-17 in heterologous cells is the co-expression of the accessory protein RTP1 or its short version RTP1S, which are also expressed by olfactory sensory neurons. Elsewhere, Saito and co-workers have proposed that this protein interacts with OR when the OR is expressed heterologously, but they have not provided evidence to prove the role of RTP1 towards OR. To investigate whether direct physical interactions between mOR256-17 and RTP1 occurs during receptor trafficking to the cell membrane, we labeled mOR256-17 and RTP1 orthogonally using the acyl carrier protein (ACP) and the peptidyl carrier protein (PCP) respectively. In addition, cyan fluorescent protein fused to the N-terminus of RTP1 enabled to study RTP1 biosynthesis in presence and absence of the mOR256-17; here we observed that a large portion of RTP1 remains in the endoplasmic reticulum while the mOR256-17 reaches the Golgi apparatus when it is co-expressed with RTP1S. Furthermore, by using two different RTP1S chimera, we could show that the cytosolic part of RTP1S is important for the mOR256-17 folding and trafficking.

EPFL2013