Barbara-Anne Battelle, Ph.D.

Biockemistry of Vision with Limulus photo

Barbara-Anne Battelle, Ph.D.
Professor of Neuroscience and Zoology

Email:battelle@whitney.ufl.edu

Retinas and photoreceptors change their function day to night. For the full range of diurnal fluctuations in visual function to occur, two things are required: rhythmic changes in environmental light and signals from internal circadian clocksWe seek to understand the biochemical mechanisms by which light and clock input, separately and together, regulate retinal and photoreceptor functions.

The horseshoe crab Limulus polyphemus, offers a number of advantages for these studies. The influence of the circadian clock on the structure and function of Limulus photoreceptors is robust and well characterized. In addition, the unique circadian organization of the Limulus visual system permits easy experimental manipulation of the circadian input to the eyes. Specifically, the circadian clock that drives changes in visual function is located in the brain, and circadian signals reach the eyes via well-characterized efferent neuronal projections through the optic nerves. The efferent projection to the lateral eye can be conveniently cut to produce an eye lacking circadian efferent input while the circadian input to the other eye, and the circadian biology of the animal as a whole, is normal.

Some years ago we showed that the biogenic amine octopamine (OA) is a transmitter released from the circadian efferent neurons and that it stimulates an increase in cAMP in Limulus photoreceptors. Much evidence suggests that OA drives circadian changes in Limulus photoreceptors by elevating cAMP and activating cAMP-dependent protein kinase (PKA).

To understand the mechanisms underlying clock driven changes in photoreceptor function, we sought to identify proteins in photoreceptors that become phosphorylated in response to clock input, OA and the activation of the cAMP cascade. We identified one such target as Limulus myosin III (LpMyo3).

Class III myosins are unusual unconventional myosins in that they have a kinase domain at their N-terminus, and as with most unconventional myosins, the functions of class III myosins are not known.

Homologues of LpMyo3 are found in the photoreceptors of Drosophila, and Drosophila mutants lacking the myosin III gene product (NINAC) undergo a light dependent degeneration. Class III myosins are also expressed in the photoreceptors of vertebrates including mammals, and in mammalian hair cells. A mutation in human Myo3A results in progressive hearing loss.

Major goals of our current research are to understand the functions of LpMyo3, how these functions are modified by phosphorylation, and how LpMyo3 phosphorylation influences photoreceptor function.

In Limulus myosin III is highly and specifically expressed in photoreceptors and it is phosphorylated by light as well as clock input. Therefore, it may be a substrate upon which light and clock signals converge.

Studies we published in 2007 combined biochemical approaches with mass spectroscopy to identify sites in LpMyo3 that are phosphorylated in vitro by cAMP and by autophosphorylation. We also identified sites that are phosphorylated in vivo in response to endogenous clock input. Interestingly, PKA autophosphorylation and clock input enhance the phosphorylation of two sites in and near loop 2 of the myosin domain, an important actin binding region. This is the first example of a myosin that becomes phosphorylated in this region. Based on studies of other myosins, the phosphorylation we observe is predicted to reduce the affinity of LpMyo3 for actin.

The association of myosins with actin is typically regulated by ATP binding to the myosin. An examination of the myosin-like properties of LpMyo3 revealed that it binds actin with the same affinity in the absence and presence of ATP and that it lacks ATPase activity. From this we conclude that LpMyo3 is not a motor, and we speculate that the affinity of LpMyo3 for actin is regulated by phosphorylation within its myosin domain instead of ATP binding. This hypothesis leads to a number of predictions which we are currently testing.

We expanded our investigations to test whether the observations we have made with LpMyo3 extend to mammalian class III myosins. One specific question we are addressing is whether the phosphorylation of loop 2 that we discovered in LpMyo3, which may be a unique mechanism for modulating actomyosin interactions, is also characteristic of mammalian class III myosins.

Vertebrates express two class III myosins that are the products of different genes (myo3A and myo3B). We developed and characterized antibodies directed against mouse Myo3A and 3B, examined the distribution of these proteins in mouse retina and have begun to examine the biochemistry of these proteins. Others have shown that unlike LpMyo3, vertebrate class III myosins are molecular motors. We have focused on examining their kinase activity and autophosphorylation sites.

Personnel

Barbara-Anne Battelle, Professor
Karen E. Kempler, Biological Scientist

Selected Publications

Battelle, B-A. (2009) Circadian rhythms in visual function in Limulus. in Comparative Aspects of Circadian Rhythm. Edited by Maria Luisa Fanjul-Moles and Raul Aguilar Roblero, Transworld Research Network 37/661 Kerala, India.

Battelle, B.A. (In press) Limulus eyes and their circadian regulation. Encyclopedia of the Eye. Elsevier.

Katti, C., Dalal, J.S., Dosé, A.C., Burnside, B. and Battelle, B-A. (In press) Cloning and distribution of Myosin 3B in the mouse retina: differential distribution in cone outer segments. Exp. Eye Research

Battelle, B.A., and Brown, Nadean L. (2008) Isolation and expression of Pax6 and atonal homologues in the American Horseshoe Crab, Limulus polyphemus. Developmental dynamics 237: 2209-2219.

Cardasis, H.L., Stevens, S.M., McClung, S., Kempler, K.E., Powell, D.H., Eyler, J.R., and Battelle, B-A. (2007) The actin-binding interface of a myosin III is phosphorylated in vivo in response to signals from a circadian clock. Biochemistry 46: 13907-13919.

Kempler, K., Toth, J., Yamashita, R., Mapel, G., Robinson, K., Cardasis, H., Stevens, S., Sellers, J.R. and Battelle, B-A. (2007) Loop 2 of Limulus myosin III is phosphorylated by protein kinase A and autophosphorylation. Biochemistry 46 : 4280-4293.

Battelle, B-A. (2006) The eyes of Limulus polyphemus (Xiphosura, Chelicerata) and their afferent and efferent projections. Arthropod Structure & Development 35:261-274.

Harzsch, S., Vilpoux, K., Blackbuirn, D.C., Platchetzki, D., Brown, N.L., Melzer, R., Kempler, K.E., Battelle, B-A. (2006) Development of the eyes and central visual pathways of the horseshoe crab Limulus polyphemus Linneaus, 1758 (Chelicerata, Xiphosura). Developmental Dynamics 225:2641-2655.

Battelle, B-A. (2006) The eyes of Limulus polyphemus (Xiphosura, Chelicerata) and their afferent and efferent projections. Arthropod Str. Devel. 35:1-14

Harzsch, S., Vilpoux, K., Blackburn, D. C., Platchetzki, D., Brown, N. L., Melzer, R., Kempler, K.E., Battelle,B-A. (2006) Evolution of arthropod visual systems: Development of the eyes and central visual pathways in the horseshoe crab Limulus polyphemus Linnaeus, 1758 (Chelicerata, Xiphosura). Devel. Dynamics. 235:2641-2655.

Harzsch, S., Wildt, M., Battelle, B., Waloszek, D. (2005) Immunohistochemical localization of neurotransmitters in the nervous system of larval Limulus polyphemus (Chelicerata, Xiphosura): evidence for a conserved protocerebral architecture in Euarthropoda. Arthropod Str. Devel. 34:327-342.

Sineschchekova, O.O., Cardasis, H.L., Severance, E.G., Smith, W.C., and Battelle, B-A. (2004) Sequential phosphorylation of visual arrestin in intact Limulus photoreceptors: Identification of a highly light-regulated site. Visual Neuroscience. 21:715-724. Dabdoub, A., Jinks, R.N., Wang, Y., Battelle, B-A. and Payne, R. (2003) Desensitization of the photoresponse by protein kinase C precedes rhabdomere disorganization and endocytosis. Visual Neuroscience 20: 241-248.

Dalal, J.S., Jinks, R.N., Cacciatore, C., Greenberg, R.M. and Battelle, B-A. (2003) Limulus opsins: diurnal regulation of expression. Visual Neuroscience 20: 523-535.

Sacunas, R.B., Papuga, M.O., Pearson, Jr., A.C., Marjanovic, M., Stroope, D.G., Weiner, W.W., Chamberlain, S.C. and Battelle, B-A. (2002) Multiple mechanisms of rhabdom shedding in the lateral eye of Limulus polyphemus. J. Comp. Neurol. 449:26-42.

Battelle, B-A. (2002) Circadian efferent input to Limulus eyes: Anatomy, circuitry and impact. Microscopy Research and Technique. 58:345-355.

Battelle, B-A. and Hart, M.K. (2002) Histamine metabolism in the visual system of the horseshoe crab Limulus polyphemus. Comparative Biochemistry and Physiology 133:135-142.

Battelle, B-A., Dabdoub, A., Malone, M.A., Andrews, A.W., Cacciatore, C., Calman, B.G., Smith, W.C., and Payne, R. (2001) Immunocytochemical localization of opsin, visual arrestin, myosin III and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors. J. Comp. Neurol. 435:211-225.

Battelle, B-A., Williams, C.D., Schremser-Berlin, J-L. and Chelsi Cacciatore. (2000). Regulation of arrestin mRNA levels in Limulus lateral eyes: Separate and combined influences of circadian efferent input and light. Visual Neuroscience 17:217-227.

Battelle, B-A., Andrews, A.W., Kempler, K.E., Edwards, S.C., and Smith, S.C. (2000). Visual arrestin in Limulus is phosphorylated at multiple sites in the light and in the dark. Visual Neuroscience 17: 813-822.

Battelle, B-A., Williams, C.D., Schremser-Berlin, J-L. and Chelsi Cacciatore (2000) Regulation of arrestin mRNA levels in Limulus lateral eyes: Separate and combined influences of circadian efferent input and light. Visual Neuroscience 17:217-227.

Battelle, B-A., B.G. Calman and M.K. Hart. (1999). Cellular distributions and functions of histamine, octopamine and serotonin in the peripheral visual system, brain and circumesophageal ring of the horseshoe crab, Limulus polyphemus. Microscopy Research and Techniques. 44:70-80.

Chen, F., Ukhanova, M., Thomas, D., Afshar, G., Tanda, S., Battelle, B-A., and Payne, R. (1999) Molecular cloning of a putative cyclic nucleotide-gated ion channel cDNA from Limulus polyphemus. J. Neurochem. 72:461-471.

Battelle, B-A., A.W. Andrews, B.G. Calman, J.R. Sellers, R.M. Greenberg and W.C. Smith. (1998). A myosin III from Limulus eyes is a clock-regulated phosphoprotein. J. Neuroscience. 18:4548-4559.

Calman, B.G., A.W. Andrews, H.M. Rissler, S.C. Edwards, and B-A. Battelle. (1996). Calcium/calmodulin-dependent protein kinase II and arrestin phosphorylation in Limulus eyes. J. Photochem. Photobiol. B: Biology. 35:33-44.

Smith, W.C., R.M. Greenberg, B.G. Calman, M.M. Hendrix, L. Hutchinson, L.A. Donoso, and B-A. Battelle. (1995). Isolation and expression of an arrestin cDNA from the horseshoe crab lateral eye. J. Neurochem. 64: 1-13.

Smith, W.C., D.A. Price, R.M. Greenberg, and B-A. Battelle. (1993). Opsins from the lateral eyes and ocelli of the horseshoe crab, Limulus polyphemus. Proc. Natl. Acad Sci. USA 90: 6150-6154