Single-cell Activity Research Articles

Population and single cell metabolic activity of UV-induced VBNC bacteria determined by CTC-FCM and D2O-labeled Raman spectroscopy

The occurrence of viable but non-culturable (VBNC) bacteria will result in significant underestimation of viable bacterial counts in drinking water. Whereas, much is unknown in characterizing their viability. In this study, two environmental isolates (Aeromonas sp. and Pseudomonas sp.) and two model strains (E. coli and S. aureus) were induced into VBNC state by UV irradiation. Then, their metabolic activity was determined by 5-cyano-2,3-ditolyl tetrazolium chloride combination flow cytometry (CTC-FCM) and D2O-labeled Raman spectroscopy, respectively, at both population and single cell levels. The results showed that almost all strains could enter VBNC state irradiated by ≥ 5 mJ/cm2 UV. When determined by CTC-FCM, the population metabolic activity for each strain did not vary significantly (p > 0.05) unless the UV dose reached 200 mJ/cm2. Their single cell activity spectrum narrowed slightly, as indicated by changes in the standard deviation of the logarithmic normal distribution (σ) of 0.015–0.033. This minute difference suggested the CTC-FCM method was suitable for assessing the essential viability of VBNC bacteria. With respect to Raman method, an obvious dose-response effect was recorded. With the UV dosages increased from 10 to 200 mJ/cm2, the CD/(CD + CH) for the four strains were reduced to between 95.7% and 47.9% of unirradiated controls, depending on strain and UV dose. Meanwhile, the single cellular Raman spectrum showed much more heterogeneously metabolic activity distribution, with some cells even entering metabolic “silence”. Considering the ubiquitous participation of water in biochemical processes, the Raman method was more appropriate in assessing the overall metabolic activity. The above findings can not only be a reference for VBNC mechanism studies, but also have the potential in optimizing disinfection and other bacterial removal processes.

The consequence of substrates of large-scale rigidity on actin network tension in adherent cells

There is compelling evidence that substrate stiffness affects cell adhesion as well as cytoskeleton organization and contractile activity. This work was designed to study the cytoskeletal contractile activity of single cells plated on micropost substrates of different stiffness using a numerical model simulating the intracellular tension of individual cells. We allowed cells to adhere onto micropost substrates of various rigidities and used experimental traction force data to infer cell contractility using a numerical model. The model shows that higher substrate stiffness leads to an increase in intracellular tension. The strength of this model is its ability to calculate the mechanical state of each cell in accordance to its individual cytoskeletal structure. This is achieved by regenerating a numerical cytoskeleton based on microscope images of the actin network of each cell. The resulting numerical structure consequently represents pulling characteristics on its environment similar to those generated by the cell in-vivo. From actin imaging we can calculate and better understand how forces are transmitted throughout the cell.

Single-cell activity tracking reveals that orbitofrontal neurons acquire and maintain a long-term memory to guide behavioral adaptation.

Learning to predict rewards based on environmental cues is essential for survival. The orbitofrontal cortex (OFC) contributes to such learning, by conveying reward-related information to brain areas such as the ventral tegmental area (VTA). Despite this, how cue-reward memory representations form in individual OFC neurons and are modified based on new information is unknown. To address this, using in vivo 2-photon calcium imaging in mice, we tracked the response evolution of thousands of OFC output neurons, including those projecting to VTA, through multiple days and stages of cue-reward learning. Collectively, we show that OFC contains several functional clusters of neurons distinctly encoding cue-reward memory representations, with only select responses routed downstream to VTA. Surprisingly, these representations were stably maintained by the same neurons even after extinction of the cue-reward pairing, and supported behavioral learning and memory. Thus, OFC neuronal activity represents a long-term cue-reward associative memory to support behavioral adaptation.

Electrocatalysis of cerium metal-organic frameworks for ratiometric electrochemical detection of telomerase activity

A ratiometric electrochemical biosensor was constructed to detect telomerase activity based on electrocatalysis of cerium-based metal-organic frameworks (CeMOFs) and conformation switch of hairpin DNA. First, the CeMOFs were synthesized using Ce as nodes and 1,3,5-benzenetricarboxylic acid as linker in a green method, and then functionalized with gold nanoparticles. The resulted Au@CeMOF tags demonstrated an excellent electrocatalysis toward hydroquinone oxidation. Meanwhile, a methylene blue (MB) modified hairpin probe was designed with telomerase primer (TP) hybridized “stem” and immobilized on the electrode surface via Au–S chemistry. In the presence of the dNTPs and telomerase, the extended TP can open the hairpin DNA and keep the MB away from the electrode surface, resulting in a decrease of electrochemical signal. In the meantime, the TP-extended part could capture the Au@CeMOF-cDNA tags on the electrode surface via hybridization, leading to the increase electrochemical signal of hydroquinone oxidation catalyzed by Au@CeMOF-cDNA tags. Thus, a ratiometric signal output mode was developed for the electrochemical detection of telomerase activity. This biosensor showed wide dynamic correlation of telomerase activity from 2 × 102 to 2 × 106 cells mL−1 with the limit of detection of 27 cells mL−1, and was applied to evaluate telomerase activity in single cell. The ratiometric electrochemical strategy based on the catalysis of MOFs provides a new avenue on signal transduction in telomerase detection.

Regulation of Neuronal Na+/K+-ATPase by Specific Protein Kinases and Protein Phosphatases.

The Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasmalemma of living cells. Numerous studies in non-neuronal tissues have shown that this transport mechanism is reversibly regulated by phosphorylation/dephosphorylation of the catalytic α subunit and/or associated proteins. In neurons, Na+/K+ transport by NKA is essential for almost all neuronal operations, consuming up to two-thirds of the neuron's energy expenditure. However, little is known about its cellular regulatory mechanisms. Here we have used an electrophysiological approach to monitor NKA transport activity in male rat hippocampal neurons in situ We report that this activity is regulated by a balance between serine/threonine phosphorylation and dephosphorylation. Phosphorylation by the protein kinases PKG and PKC inhibits NKA activity, whereas dephosphorylation by the protein phosphatases PP-1 and PP-2B (calcineurin) reverses this effect. Given that these kinases and phosphatases serve as downstream effectors in key neuronal signaling pathways, they may mediate the coupling of primary messengers, such as neurotransmitters, hormones, and growth factors, to the NKAs, through which multiple brain functions can be regulated or dysregulated.SIGNIFICANCE STATEMENT The Na+/K+-ATPase (NKA), known as the "Na+ pump," is a ubiquitous membrane-bound enzyme responsible for generating and maintaining the Na+ and K+ electrochemical gradients across the plasma membrane of living cells. In neurons, as in most types of cells, the NKA generates the negative resting membrane potential, which is the basis for almost all aspects of cellular function. Here we used an electrophysiological approach to monitor physiological NKA transport activity in single hippocampal pyramidal cells in situ We have found that neuronal NKA activity is oppositely regulated by phosphorylation and dephosphorylation, and we have identified the main protein kinases and phosphatases mediating this regulation. This fundamental form of NKA regulation likely plays a role in multiple brain functions.

High Growth Potential of Long-Term Starved Deep Ocean Opportunistic Heterotrophic Bacteria.

Experiments with bacteria in culture have shown that they often display “feast and famine” strategies that allow them to respond with fast growth upon pulses in resource availability, and enter a growth-arrest state when resources are limiting. Although feast responses have been observed in natural communities upon enrichment, it is unknown whether this blooming ability is maintained after long periods of starvation, particularly in systems that are energy limited like the bathypelagic ocean. Here we combined bulk and single-cell activity measurements with 16S rRNA gene amplicon sequencing to explore the response of a bathypelagic community, that had been starved for 1.6 years, to a sudden organic carbon supply. We observed a dramatic change in activity within 30 h, with leucine incorporation rates increasing over two orders of magnitude and the number of translationally active cells (mostly Gammaproteobacteria) increasing 4-fold. The feast response was driven by a single operational taxonomic unit (OTU) affiliated with the Marinobacter genus, which had remained rare during 7 months of starvation. Our work suggests that bathypelagic communities harbor a seed bank of highly persistent and resourceful “feast and famine” strategists that might disproportionally contribute to carbon fluxes through fast responses to occasional pulses of organic matter.

Internal representation of hippocampal neuronal population spans a time-distance continuum

The hippocampus plays a critical role in episodic memory: the sequential representation of visited places and experienced events. This function is mirrored by hippocampal activity that self organizes into sequences of neuronal activation that integrate spatiotemporal information. What are the underlying mechanisms of such integration is still unknown. Single cell activity was recently shown to combine time and distance information; however, it remains unknown whether a degree of tuning between space and time can be defined at the network level. Here, combining daily calcium imaging of CA1 sequence dynamics in running head-fixed mice and network modeling, we show that CA1 network activity tends to represent a specific combination of space and time at any given moment, and that the degree of tuning can shift within a continuum from 1 day to the next. Our computational model shows that this shift in tuning can happen under the control of the external drive power. We propose that extrinsic global inputs shape the nature of spatiotemporal integration in the hippocampus at the population level depending on the task at hand, a hypothesis which may guide future experimental studies.

In Vivo Submillisecond Two-Photon Optogenetics with Temporally Focused Patterned Light.

To better examine circuit mechanisms underlying perception and behavior, researchers need tools to enable temporally precise control of action-potential generation of individual cells from neuronal ensembles. Here we demonstrate that such precision can be achieved with two-photon (2P) temporally focused computer-generated holography to control neuronal excitability at the supragranular layers of anesthetized and awake visual cortex in both male and female mice. Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing any of the three opsins ReaChR, CoChR, or ChrimsonR, we investigated the dependence of spiking activity on the opsin's channel kinetics. We found that in all cases the use of brief illumination (≤10 ms) induces spikes of millisecond temporal resolution and submillisecond precision, which were preserved upon repetitive illuminations up to tens of hertz. To reach high temporal precision, we used a large illumination spot covering the entire cell body and an amplified laser at high peak power and low excitation intensity (on average ≤0.2 mW/μm2), thus minimizing the risk for nonlinear photodamage effects. Finally, by combining 2P holographic excitation with electrophysiological recordings and calcium imaging using GCaMP6s, we investigated the factors, including illumination shape and intensity, opsin distribution in the target cell, and cell morphology, which affect the spatial selectivity of single-cell and multicell holographic activation. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should make it easier to investigate neuronal connections and find further links between connectivity, microcircuit dynamics, and brain functions.SIGNIFICANCE STATEMENT Recent developments in the field of optogenetics has enabled researchers to probe the neuronal microcircuit with light by optically actuating genetically encoded light-sensitive opsins expressed in the target cells. Here, we applied holographic light shaping and temporal focusing to simultaneously deliver axially confined holographic patterns to opsin-positive cells in the living mouse cortex. Parallel illumination efficiently induced action potentials with high temporal resolution and precision for three opsins of different kinetics. We extended the parallel optogenetic activation at low intensity to multiple neurons and concurrently monitored their calcium dynamics. These results demonstrate fast and temporally precise in vivo control of a neuronal subpopulation, opening new opportunities for revealing circuit mechanisms underlying brain functions.

20 Years of Secretagogin: Exocytosis and Beyond.

Calcium is one of the most important signaling factors in mammalian cells. Specific temporal and spatial calcium signals underlie fundamental processes such as cell growth, development, circadian rhythms, neurotransmission, hormonal actions and apoptosis. In order to translate calcium signals into cellular processes a vast number of proteins bind this ion with affinities from the nanomolar to millimolar range. Using classical biochemical methods an impressing number of calcium binding proteins (CBPs) have been discovered since the late 1960s, some of which are expressed ubiquitously, others are more restricted to specific cell types. In the nervous system expression patterns of different CBPs have been used to discern different neuronal cell populations, especially before advanced methods like single-cell transcriptomics and activity recording were available to define neuronal identity. However, understanding CBPs and their interacting proteins is still of central interest. The post-genomic era has coined the term “calciomics,” to describe a whole new research field, that engages in the identification and characterization of CBPs and their interactome. Secretagogin is a CBP, that was discovered 20 years ago in the pancreas. Consecutively it was found also in other organs including the nervous system, with characteristic expression patterns mostly forming cell clusters. Its regional expression and subcellular location together with the identification of protein interaction partners implicated, that secretagogin has a central role in hormone secretion. Meanwhile, with the help of modern proteomics a large number of actual and putative interacting proteins has been identified, that allow to anticipate a much more complex role of secretagogin in developing and adult neuronal cells. Here, we review recent findings that appear like puzzle stones of a greater picture.

A Platform for Investigating Nuclear Organization and its Changes During Human iPSC Differentiation

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to understand the principles by which cells reorganize during the cell cycle and differentiation. We have developed a pipeline to generate high-replicate, dynamic image data on cell organization and activities using endogenous fluorescently tagged human induced pluripotent stem cells (hiPSCs) (the Allen Cell Collection at www.allencell.org). Each of the ∼25 lines express a monoallelic EGFP-tagged protein representing a cellular structure. For each structure, we take advantage of thousands of replicate high-resolution 3D images to develop quantitative image-based assays, segmentations, analyses, and computational models. We are initiating an analogous study of the nucleus, with a goal of creating a “state space of nuclear signatures.” We are endogenously tagging an initial set of eight key nuclear landmark proteins with EGFP and analyzing their sub-nuclear localization as the cells progress through the cell cycle and differentiate into cardiomyocytes. These landmarks include the nucleolus, nuclear lamina, nuclear pores, cohesin, CTCF, histones, and heterochromatin. We are also developing methods to quantify the change in localization patterns for these nuclear landmark proteins in the undifferentiated hiPSCs vs. cardiomyocytes derived from them. Preliminary image analyses, for example, reveal that H2B in cardiomyocytes displays less variation in both signal intensity and local spatial structure, generating a “smoother” protein distribution pattern within the nucleus and suggesting that our analysis approaches can detect changes in localization patterns of nuclear landmark proteins. We are also using single-cell RNAseq, FISH, and single-cell activity assays to conjoin gene expression with organization and phenotype. These cell lines and our analyses will further be integrated with genome-wide assays of chromatin architecture together with the 4D Nucleome Project and once fully characterized will be distributed publicly.

Photostimulation for In Vitro Optogenetics with High-Power Blue Organic Light-Emitting Diodes.

Optogenetics, photostimulation of neural tissues rendered sensitive to light, is widely used in neuroscience to modulate the electrical excitability of neurons. For effective optical excitation of neurons, light wavelength and power density must fit with the expression levels and biophysical properties of the genetically encoded light-sensitive ion channels used to confer light sensitivity on cells-most commonly, channelrhodopsins (ChRs). As light sources, organic light-emitting diodes (OLEDs) offer attractive properties for miniaturized implantable devices for in vivo optical stimulation, but they do not yet operate routinely at the optical powers required for optogenetics. Here, OLEDs with doped charge transport layers are demonstrated that deliver blue light with good stability over millions of pulses, at powers sufficient to activate the ChR, CheRiff when expressed in cultured primary neurons, allowing live cell imaging of neural activity with the red genetically encoded calcium indicator, jRCaMP1a. Intracellular calcium responses scale with the radiant flux of OLED emission, when varied through changes in the current density, number of pulses, frequency, and pulse width delivered to the devices. The reported optimization and characterization of high-power OLEDs are foundational for the development of miniaturized OLEDs with thin-layer encapsulation on bioimplantable devices to allow single-cell activation in vivo.

Enzymatic activity in single cells.

With the increasing recognition of the importance in addressing cell-to-cell variations for the understanding of complex biological systems, single cell analyses are becoming increasingly important. Presented in this chapter is a highly sensitive approach capable of measuring human topoisomerase 1 (TOP1) activity in single CD133 positive DLD-1 cells. The method termed On-Slide "Rolling circle Enhanced Enzyme Activity Detection (REEAD)" relies on the specific capture and lysis of CD133 positive cells on glass slides dual functionalized with anti-CD133 antibodies and a specific DNA primer. The On-Slide REEAD was demonstrated to be directly quantitative. Furthermore, the method allowed for the highly sensitive detection of TOP1 activity in single CD133 positive DLD-1 cells. The described protocol is expected to open for new possibilities in the single cell research, particularly for the investigations of chemoresistance of the cancer stem cells.

Nanokits for the electrochemical quantification of enzyme activity in single living cells.

The development of more intricate devices for the analysis of enzyme activity in single cells, and even in individual intracellular compartments, would advance the knowledge of cellular heterogeneity and protein function at subcellular locations. This chapter describes construction and implementation of nano-capillary electrodes kits, named as "Nanokits," for unprecedented cost-effective analysis of enzyme activity within single living cells and even at single lysosomes. The nanokit that is specific for the target enzyme is assembled in a nanometer-sized capillary with a working electrode, and electrochemically loaded into the cell allowing the electrochemical quantification of the enzyme activity. Furthermore, the enzyme activity in individual intracellular compartments can be characterized by reversed electrochemical pumping to confine the targeted organelle in the nano-capillary tip with the nanokit. The use of commercially available reagent kits marketed for cell population studies permits direct application of the nano-capillary electrode for targeting a variety of enzymes in single cells. This protocol will likely spark expansion in the number of groups embarking on enzyme activity investigations by clearly providing a cookbook description of new cost-effective technology for single cell analysis.

"Fix and assay": separating in-cellulo sphingolipid reactions from analytical assay in time and space using an aldehyde-based fixative.

Chemical cytometry using capillary electrophoresis (CE) is a powerful tool for measuring single-cell enzyme activity. However, these measurements are often confounding as dynamic processes within cells rapidly change depending on environment, meaning that cell handling, transport, and storage can affect signaling pathways and alter results. To meet these challenges, we describe a method utilizing aldehyde fixation to simultaneously terminate cellular reactions across a population, freezing reaction results in time prior to analytical analysis. Fluorescent sphingosine was loaded into cells of different lineages (leukemia and lymphoma cell lines and primary leukemia cells) and allowed to react before fixing. The remaining sphingosine and any products formed were then quantified with chemical cytometry utilizing CE. When cells were loaded with sphingosine followed by glyoxal fixation and immediate analysis, 55 ± 5% of lipid was recoverable compared to an unfixed control. Storage of fixed cells for 24 h showed no statistical differences in total amount of recoverable sphingolipid compared to samples analyzed immediately after fixation-though there was a difference in recovery of low-abundance products. Sphingosine kinase activity decreased in response to inhibitor treatment compared to treatment with a DMSO vehicle (21 ± 3% product formed in inhibitor-treated cells vs. 57 ± 2% in control cells), which was mirrored in single-cell measurements. This "fix and assay" strategy enables measurement of sphingosine kinase activity in single cells followed by subsequent analytical assay separated in space and time from reaction initiation, enabling greater temporal control over intracellular reactions and improving future compatibility with clinical workflow.

Analysing single live cells by scanning electrochemical microscopy.

Single live cell analysis methods provide information on the characteristics of individual cells, yielding not only bulk population averages but also their heterogeneity. Scanning electrochemical microscopy (SECM) offers single live cell activities along its topography with high accuracy probe tip positioning. Both intracellular and extracellular processes can be electrochemically examined through the use of SECM. This non-invasive technique allows for high resolution mapping of electrochemical measurements in or around the cell sample of interest. Reactive oxygen species and reactive nitrogen species can be determined in a non-invasive label-free method and utilized as a probe for cellular pathology and physiology. Membrane permeability and rate of membrane species transport can be quantified in SECM. The cell response to external stressors can be monitored and modelled. SECM is able to offer nanoscale mapping and low concentration detection, providing a powerful bioanalytical tool for live cell studies. Herein we present an overview of recent progress in the imaging and characterization of single live cells using SECM.

Single-cell proteolytic activity measurement using microfluidics for rare cell populations.

Proteolytic degradation of the extracellular matrix represents a key step in cancer dissemination and metastasis. To probe cellular proteolytic activity, fluorescent sensing substrate was developed, yet prior studies focused on average activity of thousands of cells. Considerable evidence suggests a specialized subset of cancer cells are driving metastasis, highlighting the value of single-cell approach to reveal cancer cellular heterogeneity. In addition, when only a small number of cells are available, single-cell analysis is required to draw a statistical conclusion. Here, we present a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small number (10-100) of cells can be individually characterized. Furthermore, the platform allows monitoring single cells at multiple time points for the investigation of dynamics in proteolytic activity. The presented platform represents a simple and reliable tool for single-cell proteolytic analysis, illuminating the heterogeneous and dynamic nature of cancer cells.

Single-cell assays using integrated continuous-flow microfluidics.

The recent maturation of continuous-flow microfluidic technologies has coincided with transformative new methods to profile single cells, including their genetic types, protein expression and enzyme activities. Continuous-flow high-throughput single-cell screening and sorting can reveal relationships across cellular phenotypes (e.g., enzyme activity and secretion) and genetic fingerprints. This technology provides unique opportunities, as well as experimental and computational challenges, for integrative approaches that can process large amounts of single-cell data. In this chapter, we discuss recent advances in integrated continuous-flow microfluidic approaches with a focus on measurements and statistical analysis of single-cell enzyme activity and their applications in quantitative biology, synthetic biology, and diagnosis.

Interacting neural ensembles in orbitofrontal cortex for social and feeding behaviour.

Categorically distinct basic drives (for example, for social versus feeding behaviour1-3) can exert potent influences on each other; such interactions are likely to have important adaptive consequences (such as appropriate regulation of feeding in the context of social hierarchies) and can become maladaptive (such as in clinical settings involving anorexia). It is known that neural systems regulating natural and adaptive caloric intake, and those regulating social behaviours, involve related circuitry4-7, butthe causal circuit mechanisms of these drive adjudications are not clear. Here we investigate the causal role in behaviour of cellular-resolution experience-specific neuronal populations in the orbitofrontal cortex, a major reward-processing hub thatcontains diverse activity-specific neuronal populations that respond differentially to various aspects of caloric intake8-13 and social stimuli14,15. We coupled genetically encoded activity imaging with thedevelopment and application of methods for optogenetic control of multiple individually defined cells, to both optically monitor and manipulate the activity of many orbitofrontal cortex neurons at the single-cell level in real time during rewarding experiences (caloric consumption and socialinteraction). We identified distinct populations within the orbitofrontal cortex that selectively responded to either caloric rewards or social stimuli, and found that activity of individually specified naturally feeding-responsive neurons was causally linked to increased feeding behaviour; this effect was selective as, by contrast, single-cell resolution activation of naturally social-responsive neurons inhibited feeding, and activation of neurons responsive to neither feeding nor social stimuli did not alter feeding behaviour. These results reveal the presence of potent cellular-level subnetworks within the orbitofrontal cortex that can be precisely engaged to bidirectionally control feeding behaviours subject to, for example, social influences.

Single-cell activity screening in microfluidic droplets.

Water-in-oil emulsion droplets can be used as microcompartments to contain single cells that can be subjected to activity assays in this format. Microfluidic devices produce droplets at > kHz rates and can be coupled to modules to, e.g., add reagents, incubate or measure analyte concentration optically (with sensitivities as low as 2nM). The range of optical assays includes fluorescence and absorbance detection and examples for the use of these technologies for ultrahigh-throughput sorting in directed evolution and functional metagenomics are described.

Functional Connectivity between Hippocampus and Lateral Septum is Affected in Very Young Alzheimer’s Transgenic Mouse Model

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β and tau proteins, which are believed to lead to neural damage that translates into brain dysfunction and cognitive deficits. Brain dysfunction can be evaluated by measuring single-neuron activity (spikes), global neural activity (local field potentials, LFPs) and the interaction between them. Considering that the dynamic interactions between the hippocampal pyramidal cells and lateral septum are important for proper structure function, we used the complete septo-hippocampal preparation from 30-day-old controls and J20-AD transgenic mice to record changes in spiking activity from the lateral septum and its relationship with LFP activity from the CA1 area. The cross-correlation analysis revealed that young J20 transgenic mice exhibit a significant reduction in coupling between lateral septum single-cell activity and neural network activity from the hippocampal CA1. Consistently, phase-lock analysis between lateral septum single-cell activity and CA1 neural network activity showed lower values in J20 transgenic mice. Similarly, the LFP- LFP coherence between CA1 and septum in the theta range showed lower values in J20 animals. Importantly, alterations were found before any detectable signs of cognitive deficits. Our data indicate that the disruption in the communication between hippocampus and rostral lateral septum is an early event in AD pathology and may contribute to the deficits observed during AD.