Department of Biological Chemistry uidance mechanisms in Prof. Michael Eisenbach Rina Barak, Anat Bahat, Gbacteria and Yaki Davidov, Leah Armon, Orna Liarzi, Peter Reuven, Gabriel Zarbiv

Signal transduction in bacterial AcCoA serving as an acetyl donor, or 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 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 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 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 is the and chemotaxis. and PLC and requires intracellular Ca intracellular requires and PLC by mediated is response the that and thermotaxis sperm human in operates hypothesize hypothesize that, oae o itra Ca internal on located Sagi, Y.,Sagi, M. Eisenbach, and S. Khan, A., Tur-Kaspa,Bahat, I., Gakamsky, publications Selected IP the that indicate part studies pharmacological (isthmus). lower Our its at the drop temperature in a of consequence ovulation a as at female’s established we difference is temperature addition a In that found cells. cumulus from the secreted chemoattractant only Fig. and thermotaxis. and chemotaxis sperm mammalian of mechanisms and molecular behavioral the revealing on and egg the the by of secreted chemoattractant identification efforts on current focused are Our 2). the (Figure to proximity egg close at functional mainly is that mechanism a short-range is chemotaxis and site, fertilization the towards tube Fallopian the in cells a long-range mechanism, guiding sperm response regulator CheY the regulator to response (2003) chemotaxis the of Binding 9, Med. Nature 149-150. tract. genital female in the mechanism navigation Apotential cells: mammalian sperm of M. (2003)Eisenbach, Thermotaxis H. and C., Breitbart, L. A., Giojalas,

2 A scheme of the female genital tract demonstrating the location of sperm thermotaxis thermotaxis sperm of location the demonstrating tract genital female the of scheme A in vivo in 3 Ca R , thermotaxis , is thermotaxis 2+ 2+ channel, channel, stores, stores, 2+ . We . Barak, R. and Eisenbach, M. (2004) Eisenbach, and R. Barak, Eisenbach, M. and Giojalas, L.C. (2006) L.C. Giojalas, M. and Eisenbach, Tur-Kaspa, M. and A., Eisenbach, Bahat, F., Sun A., Girsh A., Gakamsky Bahat M. (2004)Eisenbach, Towards Barak, R., Prasad, K., Shainskaya, K., Shainskaya, R., Prasad, Barak, response regulator in chemotaxis of of in chemotaxis regulator response CheY, of phosphorylation and a acetylation of Co-regulation coli unpaved road to the egg. Nature Rev. Nature egg. the to road unpaved —an in mammals guidance Sperm 2118-2121. 20, Hum. Reprod. oviduct. rabbit the within difference temperature in increase I. (2005) Periovulatory 20,Hum. Reprod. 761-767. chemoattractants. sperm secrete cells cumulus surrounding its and oocyte the Both chemotaxis: sperm M. (2005) Human Eisenbach, I. and C., N., Tur-Kaspa,E., L. Giojalas Katz Escherichia coli. Escherichia from synthetase CoA CheY regulator by acetyl- response chemotaxis the of (2004) Acetylation M. J. Eisenbach, A., Wolfe, and A. 25867-25871. 278, J. Chem. Biol. cooperativity. of Lack motor: flagellar bacterial the of complex switch intact isolated, 375-381. Gen. Physiol.Gen. 124, 105-108. J. chemotaxis. of sperm mechanism molecular the understanding . J. 342, Biol. Mol. 383-401. J. 342, Biol. Mol. Escherichia Escherichia Yan, J., R., O., Liarzi, Barak, E., Caplan, Schechtman, Gakamsky, A, J. R., and Kipnis, Oren-Benaroya, M. (2007) Ahitchhiker’s Eisenbach, R., Yan,Barak, J., and A. Shainskaya, Cohen-Ben-Lulu, G.N., N.R., Francis, Cohen-Ben-Lulu, for Biomembrane Research Biomembrane for Center Minerva Cohn the and Medicine Molecular of Institute Benoziyo support INTERNAL Foundation Minerva and Foundation, Science Binational Foundation, Science Israel the from support External in Biochemistry. Chair Professorial Djanogly Simon and Jack the of incumbent M.E. an is Acknowledgements (2008) M. Eisenbach, and A. Shainskaya, Int. J. Dev.guidance. In Biol. press. mammalian sperm to — application small is cells responsive of fraction the when chemotaxis of Analysis M. (2008) Eisenbach, and S.R. 2947-2955. 22, Hum. Reprod. by macrophages. spermatozoa human post-capacitated of M. (2007) Phagocytosis Eisenbach, Physiol. 231,Cell. 574-580. J. in chemotaxis. changes conceptual and advances through guide 251-265. J. 359, Biol. Mol. own acetylation. its CheY catalyze regulator can response M. (2006) chemotaxis The Eisenbach, 7, Biol. Cell. Mol. 276-285.. EMBO J. 27,EMBO 1134-1144. complex. more getting is complex switch flagellar (2008) bacterial The M. Eisenbach, and R.M. Johnstone, K., Y., Sagi, Prassad, G., Cecchini, Shimoni, E., Noy, D., Davidov, Y., 1260-1271. of of in chemotaxis regulator a response

Escherichia coli Escherichia In vivo In acetylation of CheY, of acetylation . J. 376, Biol. Mol.

Life Science Open Day ∙ 2008 ∙ Weizmann Institute of Science ii