Microfluidic applications in C. elegans learning and memory, reproductive aging and protein trafficking dynamics

Abstract

C. elegans has been a very good model for studying aging-related problems because it has a short life span, transparent body and 80% of its proteins have human orthologs. The conventional approach of studying C. elegans is to culture and study them on agar plates, but this has many limitations. For example, it is difficult to study a single worm’s behavior, to immobilize a worm, and to prevent worms from burrowing into the agar.

Microfluidics has been playing an important role in the current biological and medical study such as cell separation, bacteria growth, and human cell culture, because of the excellent biocompatibility of polydimethylsiloxane (PDMS), the workhorse material for microfluidic structures. The research work presented in this thesis is intended to apply microfluidic approaches to C. elegans aging study. To be specific, this thesis includes microfluidic and optic approaches that I have developed to study C.elegans’ behavior, immobilization, and long-term culture --- three categories that are most important in current research on the C. elegans using microfluidics approaches. In particular, I will explain how to use microfluidic approaches to study two important aging phenotypes: cognitive ability decline and reproductive ability decline, both of which are also two early and notable aging phenotypes in human beings. In addition, I will also discuss the application of microfluidic approaches in the study of two important transcriptional factors related to one of the most famous aging pathways in C. elegans --- the insulin/IGF-1 signaling pathway.

The first part is an introduction into existing research on C. elegans using microfluidic approaches and why researchers use C. elegans as a model organism to study aging.

The second part is on the development of a two-layer microfluidic device and the application of this device in training C. elegans in order to build a positive association between food and an odor. This device is designed not only for training the worms, but also for testing the worms’ learning and memory. Moreover, I will discuss the advantages of these microfluidic approaches compared to the conventional plate assay in details.

The third part is about microfluidic applications in C. elegans’ reproductive aging. I developed a microfluidic assay on a multiple-camera imaging platform with a real-time, automatic progeny counting system that records progeny information from many individual C. elegans hermaphrodites. This system displays many advantages over conventional plate assays, because it provides a high-time-resolution and high-throughput approach to record progeny information and thus offers researchers an efficient and labor-free technique to study reproductive aging. The main mechanism works as follows. In the current design, one device contains as many as 16 individual chambers where mother worms stay. The flow of non-proliferating bacteria not only feeds the worms but also flushes the just-hatched young progeny through a filter that separates mothers from their offspring. The progeny, which are flushed out of the chambers, are imaged by the cameras on the imaging platform and the worm signal is detected by a novel algorithm. I will also show examples of real-time progeny production information for wild-type (N2) and daf-2 (insulin receptor) mutants. In addition, I enclose the operation procedure of this microfluidic system.

The fourth part part is the study of protein trafficking dynamics. I will discuss the design and fabrication of a U-shaped microfluidic device for immobilizing worms in order to study protein trafficking dynamics over a long period of time. Specifically, I will use this device to study the trafficking dynamics between nucleus and cytoplasm of two important transcription factors DAF-16 and PQM-1. I find that in different tissues of C. elegans, or even in different cells of the same tissue, the protein traffics in and out of the nucleus in a certain sequence, which has not been reported before. This experiment discovers the heterogeneity and relationship of different cells and tissues in an organism.

In the last part, I conclude and provide an outlook for future studies.