Endoplasmic reticulum-plasma membrane contact gradients direct cell migration.

Directed cell migration is driven by the front-back polarization of intracellular signalling1-3. Receptor tyrosine kinases and other inputs activate local signals that trigger membrane protrusions at the front2,4-6. Equally important is a long-range inhibitory mechanism that suppresses signalling at the back to prevent the formation of multiple fronts7-9. However, the identity of this mechanism is unknown. Here we report that endoplasmic reticulum-plasma membrane (ER-PM) contact sites are polarized in single and collectively migrating cells. The increased density of these ER-PM contacts at the back provides the ER-resident PTP1B phosphatase more access to PM substrates, which confines receptor signalling to the front and directs cell migration. Polarization of the ER-PM contacts is due to microtubule-regulated polarization of the ER, with more RTN4-rich curved ER at the front and more CLIMP63-rich flattened ER at the back. The resulting ER curvature gradient leads to small and unstable ER-PM contacts only at the front. These contacts flow backwards and grow to large and stable contacts at the back to form the front-back ER-PM contact gradient. Together, our study suggests that the structural polarity mediated by ER-PM contact gradients polarizes cell signalling, directs cell migration and prolongs cell migration.

Rate thresholds in cell signaling have functional and phenotypic consequences in non-linear time-dependent environments.

All cells employ signal transduction pathways to respond to physiologically relevant extracellular cytokines, stressors, nutrient levels, hormones, morphogens, and other stimuli that vary in concentration and rate in healthy and diseased states. A central unsolved fundamental question in cell signaling is whether and how cells sense and integrate information conveyed by changes in the rate of extracellular stimuli concentrations, in addition to the absolute difference in concentration. We propose that different environmental changes over time influence cell behavior in addition to different signaling molecules or different genetic backgrounds. However, most current biomedical research focuses on acute environmental changes and does not consider how cells respond to environments that change slowly over time. As an example of such environmental change, we review cell sensitivity to environmental rate changes, including the novel mechanism of rate threshold. A rate threshold is defined as a threshold in the rate of change in the environment in which a rate value below the threshold does not activate signaling and a rate value above the threshold leads to signal activation. We reviewed p38/Hog1 osmotic stress signaling in yeast, chemotaxis and stress response in bacteria, cyclic adenosine monophosphate signaling in Amoebae, growth factors signaling in mammalian cells, morphogen dynamics during development, temporal dynamics of glucose and insulin signaling, and spatio-temproral stressors in the kidney. These reviewed examples from the literature indicate that rate thresholds are widespread and an underappreciated fundamental property of cell signaling. Finally, by studying cells in non-linear environments, we outline future directions to understand cell physiology better in normal and pathophysiological conditions.

Kinetics of osmotic stress regulate a cell fate switch of cell survival.

Exposure of cells to diverse types of stressful environments differentially regulates cell fate. Although many types of stresses causing this differential regulation are known, it is unknown how changes over time of the same stressor regulate cell fate. Changes in extracellular osmolarity are critically involved in physiological and pathophysiological processes in several tissues. We observe that human cells survive gradual but not acute hyperosmotic stress. We find that stress, caspase, and apoptosis signaling do not activate during gradual stress in contrast to acute treatments. Contrary to the current paradigm, we see a substantial accumulation of proline in cells treated with gradual but not acute stresses. We show that proline can protect cells from hyperosmotic stress similar to the osmoprotection in plants and bacteria. Our studies found a cell fate switch that enables cells to survive gradually changing stress environments by preventing caspase activation and protect cells through proline accumulation.

A rate threshold mechanism regulates MAPK stress signaling and survival.

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

Automated cell boundary and 3D nuclear segmentation of cells in suspension.

To characterize cell types, cellular functions and intracellular processes, an understanding of the differences between individual cells is required. Although microscopy approaches have made tremendous progress in imaging cells in different contexts, the analysis of these imaging data sets is a long-standing, unsolved problem. The few robust cell segmentation approaches that exist often rely on multiple cellular markers and complex time-consuming image analysis. Recently developed deep learning approaches can address some of these challenges, but they require tremendous amounts of data and well-curated reference data sets for algorithm training. We propose an alternative experimental and computational approach, called CellDissect, in which we first optimize specimen preparation and data acquisition prior to image processing to generate high quality images that are easier to analyze computationally. By focusing on fixed suspension and dissociated adherent cells, CellDissect relies only on widefield images to identify cell boundaries and nuclear staining to automatically segment cells in two dimensions and nuclei in three dimensions. This segmentation can be performed on a desktop computer or a computing cluster for higher throughput. We compare and evaluate the accuracy of different nuclear segmentation approaches against manual expert cell segmentation for different cell lines acquired with different imaging modalities.

Generating kinetic environments to study dynamic cellular processes in single cells.

Cells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization (smFISH). Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.

The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast.

Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1 Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1 Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them.