Vinita A Hajeri et al. (2005) International Worm Meeting "Subcellular responses to anoxia-induced suspended animation in C. elegans"

Vinita A Hajeri, Pamela A Padilla, Jesus Trejo

[International Worm Meeting, 2005]

While several animal model systems are used to understand the physiological response organisms have to oxygen deprivation, the subcellular response to oxygen deprivation, in developing embryos, is less understood. We are using the C. elegans embryo to study the molecular response to oxygen deprivation. C. elegans exposed to anoxia (<.001 kPa; 0% O2) enter into a state of suspended animation in which developmental and cell cycle progression reversibly arrests. To further characterize the subcellular response to anoxia we analyzed nuclear structures in blastomeres of embryos exposed to brief as well as prolonged periods of anoxia. Embryos were exposed to various periods of anoxia (30 minutes, 6, 12, 24 and 72 hours) and nuclear components such as chromatin structure, nuclear pore proteins, histone modifications, microtubules and the kinetochore were analyzed. Embryos exposed to at least 30 minutes of anoxia contain blastomeres that arrest at interphase, prophase, prometaphase and metaphase but not anaphase. Prophase and interphase blastomeres, of embryos exposed to 30 minutes of anoxia, contain chromatin in close proximity to the nuclear envelope, suggesting that an early subcellular response to anoxia is the nuclear reorganization of chromatin structure. The spindle checkpoint protein SAN-1, which is required for anoxia survival, localizes to the kinetochore in wild type embryos exposed to 30 minutes of anoxia. The detection of SAN-1 at the kinetochore is greatly reduced in metaphase blastomeres of embryos exposed to 24 or 72 hours of anoxia. The san-1 (RNAi) embryos exposed to 30 minutes of anoxia contain blastomeres with anaphase bridges and abnormal nuclei. This data supports the idea that the spindle checkpoint activity of SAN-1 is at the kinetochore, SAN-1 is required for the early response to anoxia, and an oxygen-sensing pathway regulates the spindle checkpoint components. In other systems the spindle checkpoint components monitor kinetochore-microtubule attachment by delaying the metaphase to anaphase transition until all chromatids are attached to microtubules. We determined that detectable depolymerization of the spindle microtubules occurs in blastomeres of embryos exposed to 6 hours of anoxia at 20<sym05>C. Spindle microtubule depolymerization was not detected in embryos exposed to short periods (30 minutes) of anoxia however, we cannot rule out the possibility that only a few, undetected, microtubules depolymerize. Identifying the early and late responses to anoxia will lead to the understanding and differentiation of mechanisms required to initiate and maintain anoxia induced suspended animation.

Reedy, A. et al. (2009) International Worm Meeting "The neural protein PKC-2 suppresses Duchenne Muscular Dystrophy."

Mariol, MC, Segalat, L., Salter, H., Lecroisey, C., Reedy, A., Gieseler, K.

[International Worm Meeting, 2009]

Duchenne muscular dystrophy (DMD) is a disease that is characterized by progressive degeneration of the skeletal, cardiac and smooth muscles, as well as neurons. DMD is caused by mutations in the Dystrophin gene, however the pathogenesis for DMD is not yet clear. Mutations in the C.elegans Dystrophin homolog (dys-1), lead to hyper-contraction, hyperactivity and an increased Acetylcholine (Ach) level. They also have infrequent body wall muscle degeneration, that can be greatly enhanced in the background of a weak mutation affecting the myogenic factor MyoD (Harte et al, 1998 ; Gieseler et al, 2000 ; comments in Chamberlain and Benian, 2000). We use this dys-1; CeMyoD double mutant as a model for DMD, as it exhibits progressive muscle degeneration. Studies in numerous model organisms, including C.elegans, indicate that loss of dystrophin or associated proteins lead to defects in Acetylcholine (Ach) transmission at the neuromuscular junction, and this may play a role in the pathophysiology of DMD. Previous genetic studies using our DMD worm model have shown that muscle degeneration is suppressed by mutations that inhibit cholinergic transmission (unc-13) or calcium influx (egl-19n582) (Mariol et al, 2001; 2007); because these mutations lead to muscle inactivity, it is impossible to conclude whether the suppression of muscle degeneration is due to muscle inactivity and/or to the suppression of another Ach induced effect. We have recently shown a mutation of pkc-2 reduces muscle degeneration in the DMD worm by almost 50%. There is currently some question as to PKC-2 localisation, and therefore the role of pkc-2 mutations in DMD pathology. A pkc-2 transgene, under a lac-Z promoter, has been shown to localise in both muscles and neurons; however, the PKC-2 antibody was observed only in neurons. (Islas-Trejo et al, 1997). GFP::pkc-2 transgenes, expressed as extrachromosomal arrays or integrated with MOSTIC have been observed in the neurons, neural cell bodies, vulva, and spermatheca, with no signal in muscles. The pkc-2 mutant was shown to have increased resistance to the Ach esterase inhibitor aldicarb, indicating it may affect Ach neurotransmission. Interestingly, pkc-2 mutants do not exhibit any obvious muscle activity phenotype. Loss of function mutations in both pkc-2 and unc-13 have been shown to suppress the DMD worm phenotype, yet no suppression was seen with RNAi. As neuronal expressed genes are relatively resistant to RNAi (Kamath et al, 2001), we think this is further evidence to suggest that the role of PKC-2 is in fact neural vs. muscular.