MPBC Faculty - Kouichi Iwasaki

Biological rhythms are intrinsic activities of organisms and are widely observed from single-cell organisms to higher vertebrates. These rhythms are thought to be part of an essential evolutionary adaptation. For example, the ~24-hour circadian rhythm is found in various organisms including humans, and emerged as an adaptation to the 24-hour day/night cycle. The range of biological rhythms is wide -- from milliseconds through years -- and includes repetitive firing of neurons (milliseconds), heartbeats (a second), breathing (seconds), internal organ peristalsis (minutes to hours), sleep cycles (hours), and ovulation cycles (weeks). Since these rhythms are intrinsic to our body physiology, it is extremely important to understand their mechanisms for medical reasons as well as for basic scientific issues.

The ultimate goal of my research is to understand the molecular and cellular mechanism of one biological rhythm, the digestive motor program (DMP) rhythm, using a model organism the nematode C. elegans. In the C. elegans experimental system, powerful genetic and molecular biological approaches are available. In particular, forward genetics has a potential to unveil new genes for various biological processes, even after the entire genome sequence is known and whole-genome assays, such as DNA microarrays, are available. Using forward genetics, new genes for various biological processes can be identified, as forward genetics relies solely on biologically relevant mutant phenotypes and not on other information such as biochemical activities and gene-expression patterns. Also, gene disruption has been very successful in the C. elegans system, for example, through the use of RNA interference (RNAi). Therefore, the feasibility of both forward and reverse genetics is a great advantage in this model system. In the C. elegans system, physiology is generally less advanced compared to other model systems. However, my lab was the first in the world to develop calcium-imaging, tissue-culture, and electrophysiological techniques specialized to the C. elegans intestine. By combining our new physiological techniques with genetic, molecular, and genomic approaches, we should be able to strongly contribute to unveiling the regulatory mechanism of the DMP rhythm and deepening our knowledge about biological rhythm physiology in general.

The digestive motor program (DMP) and Ca++ wave oscillation
In C. elegans, the DMP has a specific rhythm: the nematode intestine moves and expels gut contents every 45 seconds. To understand the genetic mechanism of the DMP rhythm, we isolated mutants with altered DMP rhythms. The mutation responsible for one of these mutants was in the inositol 1,4,5-triphosphate (IP3) receptor gene. As inositol 1,4,5-triphosphate (IP3) receptor releases Ca++ from the endoplasmic reticulum (ER) to cytoplasm and plays a central role in intracellular Ca++ signal transduction, this finding suggests that the DMP rhythm is generated by an intracellular calcium oscillation.

We developed a Ca++-imaging system for the C. elegans intestine using the calcium indicator protein Cameleon. A Ca++ influx was observed at the time of each DMP (Fig. 1). This Ca++ influx was initiated at the posterior end of the intestine and propagated at the speed of 350 micrometers/second through the anterior end (Fig. 2).We seek to understand 1) what molecular mechanism generates the Ca++ waves at the posterior intestine, 2) what mechanism propagates Ca++ waves through the intestine, and 3) what mechanism maintains the oscillation rhythm.

Auto-oscillation of the intestinal membrane potential
In various cell types of many other species, the membrane potential oscillation has been observed in correlation with Ca++ release from the endoplasmic reticulum (ER). Using the C. elegans intestine, we have found that the membrane potential also oscillates with 30-60 second cycles (Fig. 3). We would like to investigate what molecular machinery is responsible for this membrane potential oscillation and how it correlates with the Ca++ wave oscillation.

The C. elegans intestine and retrograde signaling
While presynaptic neurons regulate activities of postsynaptic cells by releasing neurotransmitters and neuropeptides, reciprocal retrograde signaling from the postsynaptic cells is essential for synaptic formation, potentiation, and plasticity, including memory formation in our central nervous system
(Fig. 4). We have found that the C. elegans intestine regulates motor neuron activities: the aex-1 gene is expressed in intestine and muscle with no expression in neurons, and changes the localization of the synaptic vesicle fusion protein UNC-13 at presynaptic terminals. The signaling molecule of this pathway appears to be a neurotrophin-like peptide, and our system should be able to elucidate this novel regulatory mechanism of presynaptic activities by non-neural cells.

Summary
Recent research in my lab has revealed very exciting aspects of a fundamental biological rhythm. Using this C. elegans system, we should be able to make significant contributions to the following important areas of biology:

1)IP3-depedent calcium oscillation and intercellular calcium wave propagation.
2)Auto-oscillation of membrane potential in non-excitable cells.
3)Retrograde control of neural activities by non-neural tissues


Kouichi Iwasaki			Assistant Professor Office Searle 5-550 (312) 503-2633 Lab Searle 5-551 (312) 503-4477 k-iwasaki@northwestern.edu
		Ph.D. Madison, WI
	Molecular Biology and Genetics of a Rhythmic Behavior Moghal, N. Garcia, L. R. Khan, L. A. Iwasaki, K. and Sternberg, P. W. Modulation of EGF receptor-mediated vulva development by the heterotrimeric G-protein Galphaq and excitable cells in C. elegans. Development in press. (2003) Doi, M. and Iwasaki, K. Regulation of retrograde signaling at neuromuscular junctions by the novel C2 domain protein AEX-1. Neuron. 33, 249-259. (2002) Iwasaki, K. and Toyonaga. R. The Rab3 GDP/GTP exchange factor homolog AEX-3 has a dual function in synaptic transmission. The EMBO Journal. 19,4806-4816.(2000) Iwasaki, K. Staunton, J. Saifee, O. Nonet, M. and Thomas J. H. aex-3 encodes a novel gene product that regulates presynaptic activity in C. elegans. Neuron 18, 613-622.(1997) Iwasaki, K. and Thomas J. H. Rhythm in Genetics. Trends in Genetics. 13, 111-115(1997). Iwasaki, K. Liu, D. W. C. and Thomas J. H. Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans. Proc Natl Acad Sci USA. 92, 10317-10321 (1995). PubMed Reference Lookup		Biological rhythms are intrinsic activities of organisms and are widely observed from single-cell organisms to higher vertebrates. These rhythms are thought to be part of an essential evolutionary adaptation. For example, the ~24-hour circadian rhythm is found in various organisms including humans, and emerged as an adaptation to the 24-hour day/night cycle. The range of biological rhythms is wide -- from milliseconds through years -- and includes repetitive firing of neurons (milliseconds), heartbeats (a second), breathing (seconds), internal organ peristalsis (minutes to hours), sleep cycles (hours), and ovulation cycles (weeks). Since these rhythms are intrinsic to our body physiology, it is extremely important to understand their mechanisms for medical reasons as well as for basic scientific issues. The ultimate goal of my research is to understand the molecular and cellular mechanism of one biological rhythm, the digestive motor program (DMP) rhythm, using a model organism the nematode C. elegans. In the C. elegans experimental system, powerful genetic and molecular biological approaches are available. In particular, forward genetics has a potential to unveil new genes for various biological processes, even after the entire genome sequence is known and whole-genome assays, such as DNA microarrays, are available. Using forward genetics, new genes for various biological processes can be identified, as forward genetics relies solely on biologically relevant mutant phenotypes and not on other information such as biochemical activities and gene-expression patterns. Also, gene disruption has been very successful in the C. elegans system, for example, through the use of RNA interference (RNAi). Therefore, the feasibility of both forward and reverse genetics is a great advantage in this model system. In the C. elegans system, physiology is generally less advanced compared to other model systems. However, my lab was the first in the world to develop calcium-imaging, tissue-culture, and electrophysiological techniques specialized to the C. elegans intestine. By combining our new physiological techniques with genetic, molecular, and genomic approaches, we should be able to strongly contribute to unveiling the regulatory mechanism of the DMP rhythm and deepening our knowledge about biological rhythm physiology in general. The digestive motor program (DMP) and Ca++ wave oscillation In C. elegans, the DMP has a specific rhythm: the nematode intestine moves and expels gut contents every 45 seconds. To understand the genetic mechanism of the DMP rhythm, we isolated mutants with altered DMP rhythms. The mutation responsible for one of these mutants was in the inositol 1,4,5-triphosphate (IP3) receptor gene. As inositol 1,4,5-triphosphate (IP3) receptor releases Ca++ from the endoplasmic reticulum (ER) to cytoplasm and plays a central role in intracellular Ca++ signal transduction, this finding suggests that the DMP rhythm is generated by an intracellular calcium oscillation. We developed a Ca++-imaging system for the C. elegans intestine using the calcium indicator protein Cameleon. A Ca++ influx was observed at the time of each DMP (Fig. 1). This Ca++ influx was initiated at the posterior end of the intestine and propagated at the speed of 350 micrometers/second through the anterior end (Fig. 2).We seek to understand 1) what molecular mechanism generates the Ca++ waves at the posterior intestine, 2) what mechanism propagates Ca++ waves through the intestine, and 3) what mechanism maintains the oscillation rhythm. Auto-oscillation of the intestinal membrane potential In various cell types of many other species, the membrane potential oscillation has been observed in correlation with Ca++ release from the endoplasmic reticulum (ER). Using the C. elegans intestine, we have found that the membrane potential also oscillates with 30-60 second cycles (Fig. 3). We would like to investigate what molecular machinery is responsible for this membrane potential oscillation and how it correlates with the Ca++ wave oscillation. The C. elegans intestine and retrograde signaling While presynaptic neurons regulate activities of postsynaptic cells by releasing neurotransmitters and neuropeptides, reciprocal retrograde signaling from the postsynaptic cells is essential for synaptic formation, potentiation, and plasticity, including memory formation in our central nervous system (Fig. 4). We have found that the C. elegans intestine regulates motor neuron activities: the aex-1 gene is expressed in intestine and muscle with no expression in neurons, and changes the localization of the synaptic vesicle fusion protein UNC-13 at presynaptic terminals. The signaling molecule of this pathway appears to be a neurotrophin-like peptide, and our system should be able to elucidate this novel regulatory mechanism of presynaptic activities by non-neural cells. Summary Recent research in my lab has revealed very exciting aspects of a fundamental biological rhythm. Using this C. elegans system, we should be able to make significant contributions to the following important areas of biology: 1)IP3-depedent calcium oscillation and intercellular calcium wave propagation. 2)Auto-oscillation of membrane potential in non-excitable cells. 3)Retrograde control of neural activities by non-neural tissues