Person:

Apfeld, Javier

This article describes the fabrication of a microfluidic device for the liquid culture of many individual nematode worms (Caenorhabditis elegans) in separate chambers. Each chamber houses a single worm from the fourth larval stage until death, and enables examination of a population of individual worms for their entire adult lifespans. Adjacent to the chambers, the device includes microfluidic worm clamps, which enable periodic, temporary immobilization of each worm. The device made it possible to track changes in body size and locomotion in individual worms throughout their lifespans. This ability to perform longitudinal measurements within the device enabled the identification of age-related phenotypic changes that correlate with lifespan in C. elegans.

The measurement of lifespan pervades aging research. Because lifespan results from complex interactions between genetic, environmental and stochastic factors, it varies widely even among isogenic individuals. The action of molecular mechanisms on lifespan is therefore visible only through their statistical effects on populations. Survival assays in C. elegans provided critical insights into evolutionarily conserved determinants of aging. To enable the rapid acquisition of survival curves at arbitrary statistical resolution, we developed a scalable imaging and analysis platform to observe nematodes over multiple weeks across square meters of agar surface at 8 μm resolution. The method generates a permanent visual record of individual deaths from which survival curves are constructed and validated, producing data consistent with the manual method for several mutants in both standard and stressful environments. Our approach allows rapid, detailed reverse-genetic and chemical screens for effects on survival and enables quantitative investigations into the statistical structure of aging.

Cells adjust their behavior in response to redox events by regulating protein activity through the reversible formation of disulfide bridges between cysteine thiols. However, the spatial and temporal control of these modifications remains poorly understood in multicellular organisms. Here, we measured the protein thiol-disulfide balance in live C. elegans using a genetically-encoded redox sensor and found that it is specific to tissues and patterned spatially within a tissue. Insulin signaling regulates the sensor's oxidation at both of these levels. Unexpectedly, we found that isogenic individuals exhibit large differences in the sensor's thiol-disulfide balance. This variation contrasts with the general view that glutathione acts as the main cellular redox buffer. Indeed, our work suggests that glutathione converts small changes in its oxidation level into large changes in its redox potential. We therefore propose that glutathione facilitates the sensitive control of the thioldisulfide balance of target proteins in response to cellular redox events.

Insulin-like peptides (ILPs) play highly conserved roles in development and physiology. Most animal genomes encode multiple ILPs. Here we identify mechanisms for how the forty Caenorhabditis elegans ILPs coordinate diverse processes, including development, reproduction, longevity and several specific stress responses. Our systematic studies identify an ILP-based combinatorial code for these phenotypes characterized by substantial functional specificity and diversity rather than global redundancy. Notably, we show that ILPs regulate each other transcriptionally, uncovering an ILP-to-ILP regulatory network that underlies the combinatorial phenotypic coding by the ILP family. Extensive analyses of genetic interactions among ILPs reveal how their signals are integrated. A combined analysis of these functional and regulatory ILP interactions identifies local genetic circuits that act in parallel and interact by crosstalk, feedback and compensation. This organization provides emergent mechanisms for phenotypic specificity and graded regulation for the combinatorial phenotypic coding we observe. Our findings also provide insights into how large hormonal networks regulate diverse traits.