Elizabeth Smith's Lab at Dartmouth College  



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Elizabeth Smith Lab - Current Projects

Below are the Specific Aims for our National Institutes of Health funded Research:

Our goal is to understand the mechanism by which calcium modulates the size and shape of ciliary and flagellar bends to control motility. This modulation must include regulation of dynein activity on specific subsets of doublet microtubules. We have demonstrated that calmodulin (CaM) is a key calcium sensor anchored to the axoneme, and that the radial spokes and central apparatus are fundamental components of the calcium control system (Smith, 2002; Wargo et al., 2004). The challenge is to determine the precise localization of CaM within the axoneme and to define the role of the central apparatus and radial spokes in CaM-mediated regulation of motility. During the previous funding period, we took advantage of the structural, functional, and biochemical approaches Chlamydomonas offers and made significance progress towards meeting this challenge. We identified three conserved complexes that exhibit calcium dependent CaM binding; two are associated with the central apparatus, and one is associated with the radial spokes (Dymek and Smith, 2007; Wargo et al., 2005). Importantly, our functional and structural studies demonstrate that these complexes play a role in controlling dynein-driven microtubule sliding (Dymek and Smith, 2007; Wargo et al., 2005; Wargo et al., 2004; Wargo and Smith, 2003). The proposed experiments are designed to test the hypothesis that the CaM-binding proteins we identified are part of a signal transduction network that alters motility in response to changes in intraflagellar calcium. Our working model is that calcium dependent CaM binding acts as a molecular switch, modifying specific protein interactions that ultimately regulate dynein-driven microtubule sliding.

Aim 1: Develop strains with defects in CaM interactors
We will employ three different strategies for generating mutant strains with defects in CaM binding proteins. Our strategies include methods for knock-out or knock-down of gene expression as well as the generation of point mutants which fail to bind CaM. We will analyze these mutants by any or a combination of the following: assessment of the phototaxis and photoshock responses, high speed motion analysis of beating flagella, analysis of dynein-driven microtubule sliding, and structural analyses using electron microscopy. These mutants will play a prominent role in testing hypotheses outlined in Aims 2 and 3.

Aim 2: Test the hypothesis that central apparatus associated CaM plays a role in modulating dynein activity on specific doublet microtubules.
We have established that the C1 tubule of the central apparatus is involved in calcium-mediated control of dynein activity on specific doublet microtubules, and have identified at least two different CaM-associated complexes which localize to C1. One complex has a higher affinity for CaM in low calcium buffer and is more thoroughly characterized. We are in the process of characterizing the second complex which has a higher affinity for CaM in high calcium. We predict that the differential localization of CaM acts as a molecular switch for calcium-induced modulation of dynein activity on specific subsets of microtubules. To test this hypothesis we will first complete our characterization of the high calcium CaM interactors. Then, we will disrupt CaM binding to each complex and test for altered dynein activity using a combined structural and functional approach.

Aim 3: Test the hypothesis that the CSC is both a structural and functional component of a signal transduction pathway that modulates dynein activity.
One CaM-complex, the CSC, localizes to the base of the radial spokes and regulates dynein-driven microtubule sliding by a mechanism that includes inner dynein arm I1 as a key target. The experiments proposed in this Aim take advantage of a combination of biochemical and functional approaches to define a mechanism for CSC regulation of dynein activity, including determining the role of calcium sensitive CaM binding. This mechanism may include altering the activity of kinases and phosphatases known to affect dynein activity. In addition, we will determine if the CSC serves as both a structural and functional link between the spokes and inner dynein arm I1. Our results will not only provide information about the regulation of specific dynein subforms but also about the assembly and targeting of this regulatory complex.

These experiments address fundamental questions about the role of calcium in regulating ciliary and flagellar motility. The discovery that calcium channels are required for sperm hyperactivation and fertility in mammals highlights the significance of intraflagellar calcium in controlling motility (Qi et al., 2007). During the next funding period we have the potential to define a molecular mechanism for CaM–mediated signal transduction that includes specific protein-protein interactions acting as switches to locally alter dynein activity. Our studies also have the broader potential to define new principles for the targeting and anchoring of molecules that define the signal transduction pathways that regulate the dynein family of motors.