Department of Biological Chemistry uidance mechanisms in Prof. Michael Eisenbach Rina Barak, Anat Bahat, Gbacteria and sperm Yaki Davidov, Leah Armon, Orna Liarzi, Peter Reuven, Gabriel Zarbiv Signal transduction in bacterial AcCoA serving as an acetyl donor, or chemotaxis by the enzyme acetyl-CoA synthetase We explore signal transduction (Acs), with acetate as the acetyl donor. strategies using chemotaxis of the In both cases the acetylation sites are bacteria Escherichia coli and Salmonella six lysine residues (Lys91, 92, 109, 119, 972 8 934 3923 typhimurium as a model. Bacterial 122 and 126), all located on the surface chemotaxis is a sophisticated system that binds to CheA, CheZ and FliM. FAX 972 8 947 2722 that integrates many different signals Mutational interference with either of [email protected] into a common output — a change in these processes results in defective www.weizmann.ac.il/~Eisenbach the direction of flagellar rotation. The chemotaxis. We even succeeded to signal transduction in E. coli chemotaxis detect CheY acetylation in vivo, finding is between two supramolecular that, on average, every molecule of complexes: the receptor supramolecular CheY is acetylated by autoacetylation. complex at the poles, and the flagellar- Our current studies are aimed at to fumarate and vice versa, mediates motor supramolecular complexes identifying the function of CheY fumarate to the switch. We established around the cell. The receptor acetylation in chemotaxis. that FRD forms a 1:1 complex with supramolecular complex includes One of the major questions in the switch-protein FliG, and that this the receptors and the enzymes that bacterial chemotaxis is how the interaction is required both for flagellar modulate the receptor activities as well switch of the bacterial flagellar motor assembly and switching the direction as the enzymes that are modulated functions. It is composed of multiple of flagellar rotation. We further by the receptors. The flagellar-motor copies of three proteins: FliM, FliG and showed that fumarate affects the supramolecular complex includes the FliN. Although some information on direction of flagellar rotation via FRD. motor and its gearbox, termed a switch. the structure of the switch proteins is This unexpected finding of functional A small protein, the excitatory response available, there is hardly any information interaction between an energy regulator CheY, shuttles back and about how the switch functions. Our conversion protein and the switch forth between the two supramolecular earlier studies identified fumarate as opens major intriguing questions, complexes and transduces sensory a switching factor, i.e., a molecule that currently addressed by us. In addition, information between them (Figure 1). enables the switch to shift from its using sophisticated single-cell imaging, Our research is focused on CheY and default state, counterclockwise, to the we study the behavior of the switch the switch. clockwise state. We used this finding as as a function of the intracellular level It is well established that the an end of a thread to investigate the of CheY. We also investigate the activity of CheY is regulated by switch function. Although our studies switching mechanism by quantitatively i its phosphorylation, the level of demonstrated that fumarate affects the studying events occurring at the switch, phosphorylation being determined switch, we could not detect its binding employing bioluminescence resonance by the histidine kinase, CheA, and to the switch complex or to individually energy transfer. the phosphatase, CheZ. We found isolated switch proteins (and to CheY, that CheY can also be activated by too). We, found, however, that the Sperm guidance in mammals acetylation. CheY acetylation can either membrane protein fumarate reductase Contrary to a prevalent belief, be carried out by autocatalysis, with (FRD), known to convert succinate there appears to be no competition in the mammalian female genital tract between large numbers of sperm cells racing towards the egg. Instead, small numbers of the ejaculated sperm cells enter the Fallopian tube and these few Weizmann Institute of ScienceWeizmann must be guided in order to make the ∙ remaining long, obstructed way to the egg. We revealed two active guidance (taxis) mechanisms: chemotaxis 2008 ∙ and thermotaxis. Both mechanisms are restricted to capacitated sperm cells, namely to cells that reached a maturation stage at which they can penetrate the egg and fertilize it. Fig. 1 Simplified scheme of signal transduction in bacterial chemotaxis of E. coli and S. Recently we found that both the egg typhimurium. Black arrows stand for regulated protein-protein interactions. CheY is a and its surrounding cumulus cells response regulator, CheA is a histidine kinase, and CheZ is a phosphatase. The scheme is secrete sperm chemoattractants, and not drawn to scale. Life Science Open Day determined that progesterone is the Mol. Cell. Biol. 7, 276-285.. Barak, R., Yan, J., Shainskaya, A. and Eisenbach, M. (2006) The chemotaxis response regulator CheY can catalyze its own acetylation. J. Mol. Biol. 359, 251-265. Eisenbach, M. (2007) A hitchhiker’s guide through advances and conceptual changes in chemotaxis. J. Cell. Physiol. 231, 574-580. Oren-Benaroya, R., Kipnis, J. and Eisenbach, M. (2007) Phagocytosis of human post-capacitated spermatozoa by macrophages. Hum. Reprod. 22, 2947-2955. Gakamsky, A, Schechtman, E., Caplan, S.R. and Eisenbach, M. (2008) Fig. 2 A scheme of the female genital tract demonstrating the location of sperm thermotaxis Analysis of chemotaxis when the and chemotaxis. fraction of responsive cells is small — application to mammalian sperm only chemoattractant secreted from isolated, intact switch complex of guidance. Int. J. Dev. Biol. In press. the cumulus cells. In addition we the bacterial flagellar motor: Lack found that a temperature difference of cooperativity. J. Biol. Chem. 278, Yan, J., Barak, R., Liarzi, O., is established at ovulation in the 25867-25871. Shainskaya, A. and Eisenbach, M. female’s oviduct as a consequence of Barak, R., Prasad, K., Shainskaya, (2008) In vivo acetylation of CheY, a temperature drop at its lower part A., Wolfe, A. J. and Eisenbach, M. a response regulator in chemotaxis (isthmus). Our pharmacological studies (2004) Acetylation of the chemotaxis of Escherichia coli. J. Mol. Biol. 376, 2+ 1260-1271. indicate that the IP3R Ca channel, response regulator CheY by acetyl- located on internal Ca2+ stores, CoA synthetase from Escherichia Cohen-Ben-Lulu, G.N., Francis, N.R., operates in human sperm thermotaxis coli. J. Mol. Biol. 342, 383-401. Shimoni, E., Noy, D., Davidov, Y., and that the response is mediated by Barak, R. and Eisenbach, M. (2004) Prassad, K., Sagi, Y., Cecchini, G., 2+ PLC and requires intracellular Ca . We Co-regulation of acetylation Johnstone, R.M. and Eisenbach, M. ii hypothesize that, in vivo, thermotaxis is and phosphorylation of CheY, a (2008) The bacterial flagellar switch a long-range mechanism, guiding sperm response regulator in chemotaxis of complex is getting more complex. cells in the Fallopian tube towards the Escherichia coli. J. Mol. Biol. 342, EMBO J. 27, 1134-1144. fertilization site, and chemotaxis is a 375-381. short-range mechanism that is mainly Eisenbach, M. (2004) Towards functional at close proximity to the understanding the molecular Acknowledgements egg (Figure 2). Our current efforts mechanism of sperm chemotaxis. J. are focused on identification of the M.E. is an incumbent of the Jack and Gen. Physiol. 124, 105-108. chemoattractant secreted by the egg Simon Djanogly Professorial Chair and on revealing the behavioral and Sun F., Bahat A., Gakamsky A., Girsh in Biochemistry. External support molecular mechanisms of mammalian E., Katz N., Giojalas L. C., Tur-Kaspa, from the Israel Science Foundation, sperm chemotaxis and thermotaxis. I. and Eisenbach, M. (2005) Human Binational Science Foundation, and sperm chemotaxis: Both the oocyte Minerva Foundation Institute of ScienceWeizmann and its surrounding cumulus cells ∙ Selected publications secrete sperm chemoattractants. INTERNAL support Bahat, A., Tur-Kaspa, I., Gakamsky, Hum. Reprod. 20, 761-767. Benoziyo Institute of Molecular 2008 A., Giojalas, L. C., Breitbart, H. and ∙ Bahat, A., Eisenbach, M. and Tur-Kaspa, Medicine and the Cohn Minerva Center Eisenbach, M. (2003) Thermotaxis of I. (2005) Periovulatory increase in for Biomembrane Research mammalian sperm cells: A potential temperature difference within the navigation mechanism in the female rabbit oviduct. Hum. Reprod. 20, genital tract. Nature Med. 9, 149-150. 2118-2121. Sagi, Y., Khan, S. and Eisenbach, M. Eisenbach, M. and Giojalas, L.C. (2006) (2003) Binding of the chemotaxis Sperm guidance in mammals — an response regulator CheY to the unpaved road to the egg. Nature Rev. Life Science Open Day.