The ability of an organism to live in various environments is dictated by interplay of environmental and genetic factors. Ongoing projects aim to explore how these factors impact an organism's homeostatic state.
Networked control of muscle protein degradation
Figure 1. Partial model of signal networks regulating protein degradation in C. elegans muscle. Far Left (Green): Caspase activation is controlled by degenerin channel contact with collagen in the extra-cellular matrix. Left (Blue): Degradation by calpains is regulated by integrin attachment to the basement membrane. Middle (Yellow): Autophagic degradation is controlled by a balance of signal from Insulin/Insulin-like Receptor (negative regulator, green lines) and autocrine Fibroblast Growth Factor signal (positive regulator, red lines). Calcium overload, signalling via CaMKII, also promotes autophagic degradation. Right (Pink): Intra-cellular calcium controlled by a combination of membrane depolarization and G-protein signalling events is required to negatively regulate proteasome-based degradation.
Human skeletal muscle is a multifunctional tissue containing up to half the total protein content of the body, a metabolic buffer that can be mobilized by protein catabolism in times of organismal need. Protein is also degraded during adaptive muscle remodelling upon exercise, but extreme degradation in diverse clinical conditions can compromise function(s) and threaten life. An extensive body of literature on muscle atrophy shows that degradation is a regulated process and identifies both extramuscular regulators (e.g., insulin, IGF-1, TNF-alpha) and intramuscular enzymes that degrade proteins (e.g., proteasomes, calpains, lysosomes). However, we lack a comprehensive understanding of the molecular signalling networks that tie extramuscular regulators to intramuscular proteases. Such information is desirable both in order to understand the degree to which the regulation of muscle atrophy is distinct in different clinical populations and to understand how an organism balances various metabolic needs to maintain homeostasis in various environments.
The soil nematode Caenorhabditis elegans has been developed into a model system for studying the regulation of muscle protein degradation. As in human muscle, proteolysis in C. elegans muscle is triggered by starvation, dietary restriction, environmental toxins, or denervation and responds to altered levels of growth factors (IGF, FGF, TGF-beta). We have begun to characterize the signalling pathways that mediate proteolysis in response to each of these physiologic alterations. Some of these pathways are interrelated while others appear independent. This work has revealed more complexity in the regulation of degradation than anticipated, with at least 150 genes known to be involved.
We are building toward a comprehensive model of the signalling networks that control muscle protein degradation. Using RNA-mediated interference (RNAi) we have examined all "muscle mutant" genes and all genes predicted to encode a protein kinase or phosphatase. We are currently functionally clustering identified genes either within established networks of regulatory signals or within newly identified pathways. In parallel, we are determining which pathways are active in human muscle and how these signalling pathways are related to adaptation to altered environmental conditions, for example decreased food, environmental toxins and spaceflight.
Biological alterations in response to spaceflight
Figure 2. C. elegans returning from the International Space Station. Our experiment on Expedition 22 (CERISE) was carried out inside KIBO module. Photo courtesy of NASA.
Spaceflight induces physiological changes across phyla with altered metabolism being universally observed. Despite decades of study, relatively little is known of how adaptation to the spaceflight environment occurs at the molecular level. Understanding adaptation to spaceflight at the molecular level is desirable both to allow pharmacological intervention to speed (or inhibit) (un)desirable adaptations and to understand how the Earth environment has shaped life.
C. elegans has been established a model system for space life sciences and astrobiology. Our efforts have involved developing automated culturing and experimentation so that these can be conducted remotely without need for astronaut intervention. We have confirmed that the culture system works in space and have established that C. elegans exhibits altered muscle physiology with gene expression and protein changes that are identical to those observed in mammalian muscles. Microarray analysis of global gene expression suggests that two signalling pathways that regulate C. elegans response to the environment (Insulin, TGF-beta) may indeed regulate C. elegans response to spaceflight. Comparative analysis with Drosophila and mouse gene expression data suggests conserved types of tissue specific molecular change. A direct demonstration of conserved mechanism(s) of adaptation is still required.
Presently we are finishing analysis of data obtained using the automated culturing system onboard the International Space Station (Expeditions 14 and 15) and during a non-automated experiment (Expedition 22). As additional spaceflight opportunities become available, we anticipate demonstrating that predicted mechanisms control spaceflight adaptation using appropriate genetic and pharmacologic tools. In the future, we anticipate developing additional tools that allow us to remotely monitor C. elegans response to the environment, for example the moon.
Negative regulation of muscle protein degradation by attachment to the basement membrane
Figure 3. Acute disruption of C. elegans muscle attachment to the basement membrane. Worms containing GFP labeled sarcomeres were subject to acute, genetic, disruption of an integrin muscle attachment complex. Normal arrayed sarcomeres can be seen in the lower left of the image while 'balls' of collapsed arrayed sarcomeres can be viewed in the middle of the image.
Our interests listed above recently combined when we discovered several the 'muscle' genes found in decreased amounts following spaceflight also appear to regulate muscle protein degradation. The proteins encoded by these genes form part of a muscle attachment complex. Proteins in this attachment complex are linked to various Limb Girdle Muscular Dystrophies as well as muscle growth and atrophy in response to exercise and immobilization. Ongoing work consists of defining the protein members of this attachment complex from among at least 150 candidates, defining the protease(s) activated in response to disruption of muscle attachment, and determining the link(s), if any, between cytosolic protein degradation, sarcomere disruption, and loss of movement. Additionally, we are testing how a particular sodium channel appears to regulate caspases activation in response to loss of attachment to the basement membrane.