Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.     

The Unwhole Organism

The existence of gutless animals was known, and their putativenutritional processes investigated for several decades, beforethe sulfide-oxidizing symbiosis that sustains them was discovered.Research into the large, gutless Pogonophora of the marine,thermal vent communities, and the relatively large, gutless,bivalved mollusc Solemya reidi provided an adequate paradigmand stimulated exploration of the evolutionary impact of thesymbiosis. These "unwhole" organisms provide an epistemologicalmodel for studying the necessity, as well as the limitationsof the concept of organism. For non-parasitic gutless animals, and for others with reducedguts, a variety of reductionistic, adaptationistic and organicistichypotheses were advanced, but despite a general familiaritywith parallel symbioses there was a reluctance to transcendthe organismic mind-set Free-living sulfide-oxidizing bacteria inhabit a two-dimensionalenvironment: the interface between aerobic and anaerobic environments.A host, such as Solemya, adds a third dimension, regulatingthe supply of necessary oxygen and sulfide at the molecular,functional morphological, and behavioural levels. Morphologicalcorrelations of the symbiosis in bivalves include expansionof gills to house bacteria, paedomorphic reduction of outerdemibranchs and palps, and reduction or loss of siphons andguts. In S. reidi symbiont transmission appears to be vertical,i.e., an intimate transferral from one generation to the next. Initial failure to realise that gutless animals are sustainedby intracellular bacteria echoes the original response to theendosymbiotic theory of the origins of eukaryotes, which hada larger historical context. Yet evolution by association hasperiodically produced major advances in the history of organisms.While simplistic reductionism has a false allure, organicismalso has limitations that are illustrated by the above casehistory. Whether we identify ourselves as adaptationistic neo-Darwinists,or require that greater emphasis be placed on the evolutionof integrated dynamic wholes, as do the structuralists, we mustsomehow accommodate the ultraorganismic evolution of new "wholes"by the association of previously independent forms.     

An Integrated In Vitro Imaging Platform for Characterizing Filarial Parasite Behavior within a Multicellular Microenvironment

Lymphatic Filariasis, a Neglected Tropical Disease, is caused by thread-like parasitic worms, including B. malayi, which migrate to the human lymphatic system following transmission. The parasites reside in collecting lymphatic vessels and lymph nodes for years, often resulting in lymphedema, elephantiasis or hydrocele. The mechanisms driving worm migration and retention within the lymphatics are currently unknown. We have developed an integrated in vitro imaging platform capable of quantifying B. malayi migration and behavior in a multicellular microenvironment relevant to the initial site of worm injection by incorporating the worm in a Polydimethylsiloxane (PDMS) microchannel in the presence of human dermal lymphatic endothelial cells (LECs) and human dermal fibroblasts (HDFs). The platform utilizes a motorized controllable microscope with CO₂ and temperature regulation to allow for worm tracking experiments with high resolution over large length and time scales. Using post-acquisition algorithms, we quantified four parameters: 1) speed, 2) thrashing intensity, 3) percentage of time spent in a given cell region and 4) persistence ratio. We demonstrated the utility of our system by quantifying these parameters for L3 B. malayi in the presence of LECs and HDFs. Speed and thrashing increased in the presence of both cell types and were altered within minutes upon exposure to the anthelmintic drug, tetramisole. The worms displayed no targeted migration towards either cell type for the time course of this study (3 hours). When cells were not present in the chamber, worm thrashing correlated directly with worm speed. However, this correlation was lost in the presence of cells. The described platform provides the ability to further study B. malayi migration and behavior.     

Multicellular sprouting in vitro

Cell motility and its guidance through cell-cell contacts is instrumental in vasculogenesis and in other developmental or pathological processes as well. During vasculogenesis, multicellular sprouts invade rapidly into avascular areas, eventually creating a polygonal pattern. Sprout elongation, in turn, depends on a continuous supply of endothelial cells, streaming along the sprout toward its tip. As long-term videomicroscopy of in vitro cell cultures reveal, cell lines such as C6 gliomas or 3T3 fibroblasts form multicellular linear arrangements in vitro, similar to the multicellular vasculogenic sprouts. We show evidence that close contact with elongated cells enhances and guides cell motility. To model the patterning process we augmented the widely used cellular Potts model with an inherently nonequilibrium interaction whereby surfaces of elongated cells become more preferred adhesion substrates than surfaces of well-spread, isotropic cells.     

纤维堆囊菌的多细胞形态发生过程

粘细菌是具有复杂多细胞行为的革蓝氏阴性细菌,纤维堆囊菌是粘细菌中唯一能够降解纤维素的粘细菌种属。本文通过扫描电子显微镜和相差光学显微镜,分析了纤维堆囊菌在纤维素基质上的生长和子实体形态发生的多细胞行为特征,给出了生长和子实体发育的模式图谱。     

Multicellular dynamics during epithelial elongation

The reorganization of multicellular populations to produce an elongated tissue structure is a conserved mechanism for shaping the body axis and several organ systems. In the Drosophila germband epithelium, this process is accompanied by the formation of a planar polarized network of junctional and cytoskeletal proteins in response to striped patterns of gene expression. Actomyosin cables and adherens junctions are dynamically remodeled during intercalation, providing the basis for polarized cell behavior. Quantitative analysis of cell behavior in living embryos reveals unexpected cell population dynamics that include the formation of multicellular rosette structures as well as local neighbor exchange.     

Evolution of the Multicellular Animals

SYNOPSIS. Molecular sequence analysis is providing new insightsinto the study of metazoan relationships. The use of ribosomalRNA sequences is revising many of the metazoan phylogenies thathave been established traditionally with anatomical and embryologicaldata. Four new findings that seem to be well supported by moleculardata, both from the authors' laboratories and from others, aredescribed and discussed. First, the arthropods are members ofa deep primary clade within the protostomes and are not thesister taxa of either the annelids or the mollusks. Second,the lophophorate animals are clearly protostomes and are containedwithin a lophotrochozoan superclade including the mollusks,annelids, and many other phyla. Third, the arthropods togetherwith all other molting animals comprise a second monophyleticsuperclade within the protostomes, the ecdysozoa. Fourth, theplatyhelminthes are contained within the lophotrochozoan superclade.