Stochastic gene expression out-of-steady-state in the cyanobacterial circadian clock

Recent advances in measuring gene expression at the single-cell level have highlighted the stochastic nature of messenger RNA and protein synthesis. Stochastic gene expression creates a source of variability in the abundance of cellular components, even among isogenic cells exposed to an identical environment. Recent integrated experimental and modelling studies have shed light on the molecular sources of this variability. However, many of these studies focus on systems that have reached a steady state and therefore do not address a large class of dynamic phenomena including oscillatory gene expression. Here we develop a general protocol for analysing and predicting stochastic gene expression in systems that never reach steady states. We use this framework to analyse experimentally stochastic expression of genes driven by the Synechococcus elongatus circadian clock. We find that, although the average expression at two points in the circadian cycle separated by 12 hours is identical, the variability at these two time points can be different. We show that this is a general feature of out-of-steady-state systems. We demonstrate how intrinsic noise sources, owing to random births and deaths of mRNAs and proteins, or extrinsic noise sources, which introduce fluctuations in rate constants, affect the cell-to-cell variability. To distinguish experimentally between these sources, we measured how the correlation between expression fluctuations of two identical genes is modulated during the circadian cycle. This quantitative framework is generally applicable to any out-of-steady-state system and will be necessary for understanding the fidelity of dynamic cellular systems.     

Progress of AFM single-cell and single-molecule morphology imaging

Atomic force microscopy (AFM) can probe single living cells and single native membrane proteins in natural fluid environments with label-free high spatial resolution. It has thus become an important tool for cellular and molecular biology that significantly complements traditional biochemical and biophysical techniques such as optical and electron microscopy and X-ray crystallog-raphy. Imaging surface topography is the primary application of AFM in the life sciences. Since the early 1990s, researchers have used AFM to investigate morphological features of living cells and native membrane proteins with impressive results. Steady improvements in AFM techniques for imaging soft biological samples have greatly expanded its applications. Based on the authors’ own research in AFM imaging of living cell morphologies, a review of sample preparation procedures for single-cell and single-molecule imaging experiments is presented, along with a summary of recent progress in AFM imaging of living cells and native membrane proteins. Finally, the challenges of AFM high-resolution imaging at the single-cell and single-molecule levels are discussed.     

癌症单细胞数据拷贝数变异检测方法

随着单细胞测序数据的异质性优势在癌症研究中的逐渐体现，现有拷贝数变异检测方法在检测单细胞数据时效果差的问题亟待解决。提出一种新的单细胞数据拷贝数变异检测方法（FL-CNV），通过动态窗口划分及数据估算对变异区间进行范围估计和断点确定，以明确拷贝数变异的断点位置和变异类型。所提方法突破了现有检测方法在单细胞数据上的局限性，对其检测效果在模拟数据和真实数据上进行了实验验证。结果表明：与现有方法相比，本文所提方法能显著提高拷贝数变异检测的精度和敏感度，且所得结果与比较基因组杂交（array-based comparative genomic hybridization，aCGH）的拷贝数变异进行了相关性验证，具有更高的可信度。     

Alterations in phosphoproteome under salt stress in Thellungiella roots

High salinity stress is a major environmental factor that limits plant’s distribution and productivity. An Arabidopsis-related halophyte, Thellungiella halophila, is an emerging model system used for plant abiotic stress tolerance research. Previous studies have suggested that protein phosphorylation has a crucial role in the high salinity response in plants. However, the phosphoproteome differential expression under high salinity stress in halophytes has not been well studied. In this report, phosphoproteome differential expression was analyzed under high salinity stress in Thellungiella roots. Twenty-six putative phosphoproteins were found to have changed expression pattern at the post-translational level. Twenty of these were identified by mass spectrometric analysis, including 18 upregulated and two downregulated phosphoproteins. These proteins were involved in a variety of cellular processes, such as signal transduction, ROS detoxification, energy pathway, protein synthesis and protein folding. While most of these salt-responsive putative phosphoproteins are known salt-stress-related proteins, some of them have not been previously reported. Our results provide not only new insights into salt stress responses in Thellungiella but also a good foundation for further investigation of these high salinity-regulated phosphoproteins.     

Variability and memory of protein levels in human cells

Protein expression is a stochastic process that leads to phenotypic variation among cells. The cell-cell distribution of protein levels in microorganisms has been well characterized but little is known about such variability in human cells. Here, we studied the variability of protein levels in human cells, as well as the temporal dynamics of this variability, and addressed whether cells with higher than average protein levels eventually have lower than average levels, and if so, over what timescale does this mixing occur. We measured fluctuations over time in the levels of 20 endogenous proteins in living human cells, tagged by the gene for yellow fluorescent protein at their chromosomal loci. We found variability with a standard deviation that ranged, for different proteins, from about 15% to 30% of the mean. Mixing between high and low levels occurred for all proteins, but the mixing time was longer than two cell generations (more than 40 h) for many proteins. We also tagged pairs of proteins with two colours, and found that the levels of proteins in the same biological pathway were far more correlated than those of proteins in different pathways. The persistent memory for protein levels that we found might underlie individuality in cell behaviour and could set a timescale needed for signals to affect fully every member of a cell population.     

Probing cellular protein complexes using single-molecule pull-down

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.     

Neuronal pacemaker for breathing visualized in vitro.

Breathing movements in mammals arise from a rhythmic pattern of neural activity, thought to originate in the pre-B?tzinger complex in the lower brainstem. The mechanisms generating the neural rhythm in this region are unknown. The central question is whether the rhythm is generated by a network of bursting pacemaker neurons coupled by excitatory synapses that synchronize pacemaker activity. Here we visualized the activity of inspiratory pacemaker neurons at single-cell and population levels with calcium-sensitive dye. We developed methods to label these neurons retrogradely with the dye in neonatal rodent brainstem slices that retain the rhythmically active respiratory network. We simultaneously used infrared structural imaging to allow patch-clamp recording from the identified neurons. After we pharmacologically blocked glutamatergic synaptic transmission, a subpopulation of inspiratory neurons continued to burst rhythmically but asynchronously. The intrinsic bursting frequency of these pacemaker neurons depended on the baseline membrane potential, providing a cellular mechanism for respiratory frequency control. These results provide evidence that the neuronal kernel for rhythm generation consists of a network of synaptically-coupled pacemaker neurons.     

HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS

A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.     

Brain-wide neuronal dynamics during motor adaptation in zebrafish

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.     

Prion protein remodelling confers an immediate phenotypic switch

In a variety of systems, proteins have been linked to processes historically limited to nucleic acids, such as infectivity and inheritance. These atypical proteins, termed prions, lack sequence homology but are collectively defined by their capacity to adopt multiple physical and therefore functional states in vivo. Newly synthesized prion protein generally adopts the form already present in the cell, and this in vivo folding bias directs the near faithful transmission of the corresponding phenotypic state. Switches between the prion and non-prion phenotypes can occur in vivo; however, the fate of existing protein during these transitions and its effects on the emergence of new traits remain major unanswered questions. Here, we determine the changes in protein-state that induce phenotypic switching for the yeast prion Sup35/[PSI(+)]. We show that the prion form does not need to be specified by an alternate misfolding pathway initiated during Sup35 synthesis but instead can be accessed by mature protein. This remodelling of protein from one stable form to another is accompanied by the loss of Sup35 activity, evoking a rapid change in cellular phenotype within a single cell cycle.     

Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.     

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.     

From molecular noise to behavioural variability in a single bacterium

The chemotaxis network that governs the motion of Escherichia coli has long been studied to gain a general understanding of signal transduction. Although this pathway is composed of just a few components, it exhibits some essential characteristics of biological complexity, such as adaptation and response to environmental signals. In studying intracellular networks, most experiments and mathematical models have assumed that network properties can be inferred from population measurements. However, this approach masks underlying temporal fluctuations of intracellular signalling events. We have inferred fundamental properties of the chemotaxis network from a noise analysis of behavioural variations in individual bacteria. Here we show that certain properties established by population measurements, such as adapted states, are not conserved at the single-cell level: for timescales ranging from seconds to several minutes, the behaviour of non-stimulated cells exhibit temporal variations much larger than the expected statistical fluctuations. We find that the signalling network itself causes this noise and identify the molecular events that produce it. Small changes in the concentration of one key network component suppress temporal behavioural variability, suggesting that such variability is a selected property of this adaptive system.     

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.     

Conformational entropy in molecular recognition by proteins

Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein conformational entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in conformational dynamics as a proxy for corresponding changes in conformational entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding conformational entropy is linearly related to the change in the overall binding entropy. This indicates that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association.