A software solution for recording circadian oscillator features in time-lapse live cell microscopy

BACKGROUND: Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. RESULTS: In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. CONCLUSIONS: Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. AVAILABILITY: CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/

Roles of human peripheral circadian system in metabolic diseases and cancer

Circadian oscillation of biological processes has been described in most of the light-sensitive organisms. There is a growing body of evidence for connection between a number of metabolic pathologies and the circadian clockwork. We have set up the experimental system for long-term recording of circadian reporter oscillations in human primary cultured cells from different tissue types, and revealed the presence of a robust circadian oscillator in human pancreatic islets and islet cells using this approach. The prevalence of obesity and diabetes in modern society is taking on enormous proportions. It is therefore of major importance to identify the molecular basis of circadian rhythmicity in human peripheral tissues in physiological conditions, and upon obesity/T2D. Furthermore, we explored the molecular makeup of human thyroid clock, and its potential roles in thyroid malignancies. This approach might bring new insights into the role of circadian clocks in thyroid cancer, and have important clinical perspectives

The mammalian circadian timing system: organization and coordination of central and peripheral clocks

Most physiology and behavior of mammalian organisms follow daily oscillations. These rhythmic processes are governed by environmental cues (e.g., fluctuations in light intensity and temperature), an internal circadian timing system, and the interaction between this timekeeping system and environmental signals. In mammals, the circadian timekeeping system has a complex architecture, composed of a central pacemaker in the brain's suprachiasmatic nuclei (SCN) and subsidiary clocks in nearly every body cell. The central clock is synchronized to geophysical time mainly via photic cues perceived by the retina and transmitted by electrical signals to SCN neurons. In turn, the SCN influences circadian physiology and behavior via neuronal and humoral cues and via the synchronization of local oscillators that are operative in the cells of most organs and tissues. Thus, some of the SCN output pathways serve as input pathways for peripheral tissues. Here we discuss knowledge acquired during the past few years on the complex structure and function of the mammalian circadian timing system

Cell-specific resetting of mouse islet cellular clocks by glucagon, glucagon-like peptide 1 and somatostatin

Molecular clocks, operative in pancreatic islet cells, represent an intrinsic mechanism regulating intracellular metabolism and hormone secretion. Glucagon, somatostatin and glucagon-like peptide 1 (GLP-1) are essential coordinators of islet physiology. Here, we assess the synchronizing capacity of glucagon, somatostatin and GLP-1 on pancreatic α- and β-cell circadian clocks

A pancreatic clock times insulin release

Circadian oscillators of beta cells control insulin secretion and glucose homeostasis [Also see Research Article by Perelis et al. ] </jats:p

Thyroid Circadian Timing: Roles in Physiology and Thyroid Malignancies

The circadian clock represents an anticipatory mechanism, well preserved in evolution. It has a critical impact on most aspects of the physiology of light-sensitive organisms. These rhythmic processes are governed by environmental cues (fluctuations in light intensity and temperature), an internal circadian timing system, and interactions between this timekeeping system and environmental signals. Endocrine body rhythms, including hypothalamic-pituitary-thyroid (HPT) axis rhythms, are tightly regulated by the circadian system. Although the circadian profiles of thyroid-releasing hormone (TRH), thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) in blood have been well described, relatively few studies have analyzed molecular mechanisms governing the circadian regulation of HPT axis function. In this review, we will discuss the latest findings in the area of complex regulation of thyroid gland function by the circadian oscillator. We will also highlight the molecular makeup of the human thyroid oscillator as well as the potential link between thyroid malignant transformation and alterations in the clockwork

Circadian timing of metabolism in animal models and humans

Most living beings, including humans, must adapt to rhythmically occurring daily changes in their environment that are generated by the Earth's rotation. In the course of evolution, these organisms have acquired an internal circadian timing system that can anticipate environmental oscillations and thereby govern their rhythmic physiology in a proactive manner. In mammals, the circadian timing system coordinates virtually all physiological processes encompassing vigilance states, metabolism, endocrine functions and cardiovascular activity. Research performed during the past two decades has established that almost every cell in the body possesses its own circadian timekeeper. The resulting clock network is organized in a hierarchical manner. A master pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, is synchronized every day to the photoperiod. In turn, the SCN determines the phase of the cellular clocks in peripheral organs through a wide variety of signalling pathways dependent on feeding cycles, body temperature rhythms, oscillating bloodborne signals and, in some organs, inputs of the peripheral nervous system. A major purpose of circadian clocks in peripheral tissues is the temporal orchestration of key metabolic processes, including food processing (metabolism and xenobiotic detoxification). Here, we review some recent findings regarding the molecular and cellular composition of the circadian timing system and discuss its implications for the temporal coordination of metabolism in health and disease. We focus primarily on metabolic disorders such as obesity and type 2 diabetes, although circadian misalignments (shiftwork or 'social jet lag') have also been associated with the aetiology of human malignancies