1. CHEMOTAXIS: Signalling the way forward Christian Westermeier

2. Hauptseminar Biophysik der Systeme: Chemotaxis2 Chemotaxis of Dictyostelium discoideum Structure of the presentation short introduction signalling pathways: - directional sensing - establishment of polarity - locomotion of the cell summarization

3. Hauptseminar Biophysik der Systeme: Chemotaxis3 Chemotaxis Some types of cells can sense the presence of extracellular signals and guide their movement in the direction of the concentration gradient

4. Hauptseminar Biophysik der Systeme: Chemotaxis4 Chemotaxis is essential for survival …if cells fail to reach their proper destinations they die, or even the organism dies  mechanisms for processing chemotactic signals must have been optimized during evolution

5. Hauptseminar Biophysik der Systeme: Chemotaxis5 Problem: Hit the Target (…or “Jack, the lonely cell”) uniform cell is attracted by a destination

6. Hauptseminar Biophysik der Systeme: Chemotaxis6 Problem: Hit the Target (…or “Jack, the lonely cell”) uniform cell is attracted by a destination ?

7. Hauptseminar Biophysik der Systeme: Chemotaxis7 Problem: Hit the Target (…or “Jack, the lonely cell”) 1. Directional Sensing

8. Hauptseminar Biophysik der Systeme: Chemotaxis8 Orientation Information: spatial  temporal Prokaryotes (1 – 2 μm)  only temporal component  „random walk“ Eukaryotes (10 – 20 μm)  can process spatial + temporal  respond to differences in chemoattractant concentration down to 2 – 10% between back and front of the cell

9. Hauptseminar Biophysik der Systeme: Chemotaxis9 Chemoattractants set directions Dicties undergo directed cell migration in response to nutrients platelet-activating-factor (PAF) cAMP

10. Hauptseminar Biophysik der Systeme: Chemotaxis10 Detection of Ligands Receptors in the membrane of the cell (4 different ones for cAMP) seven-transmembrane-spanning receptors couple to heterotrimeric G proteins + also directly to downstream events (G-protein independent)

11. Hauptseminar Biophysik der Systeme: Chemotaxis11 G proteins in Dicties complex of α-,β-,γ-subunit only one β-subunit and one γ-subunit exist βγ-complex essential for chemotaxis 11 different α-subunits (45% identical) Gα 2 mainly linked to cAMP-receptors

12. Hauptseminar Biophysik der Systeme: Chemotaxis12 Receptors coupled to G-proteins Bild

13. Hauptseminar Biophysik der Systeme: Chemotaxis13 Localization of receptors even in chemotaxing Dicties the chemoattractant receptors are uniformly distributed along the cell, and also the G protein subunits. There are no specific localizations in these cells.  remaining distribution allows the cell to respond rapidly to gradient changes along the length of the cell: no modulation of receptor affinity  external gradient of chemoattractant ~receptor occupancy ~G-protein activation

14. Hauptseminar Biophysik der Systeme: Chemotaxis14 Problem: Hit the Target (…or “Jack, the lonely cell”) 2. Polarization

15. Hauptseminar Biophysik der Systeme: Chemotaxis15 Polarity response to a detected spatial gradient: extension of the cell membrane  pseudopod formation at the leading edge + suppression of lateral pseudopods elsewhere + retraction of the uropod at the rear of the cell

16. Hauptseminar Biophysik der Systeme: Chemotaxis16 absence of chemoattractants unpolarized dicti cells extend pseudopodia in random directions pseudopodia are periodically induced at the cell periphery and last for ~60 sec until they are retracted also more then 1 pseudopod at the same time properties of pseudopodia are almost independent of presence of chemoattractants  self-organizing structures (?)  simplifies triggering + integration all over the cell

17. Hauptseminar Biophysik der Systeme: Chemotaxis17 presence of chemoattractants biochemical + functional polarization front of the cell (leading edge): contains actin filaments  induce formation of new pseudopodia (only one at a time) back of the cell (uropod): contains myosin filaments  retracting the uropod

18. Hauptseminar Biophysik der Systeme: Chemotaxis18 Phosphoinositide signalling PIP 2 PIP 3 Phosphatidylinositol-3,4,5-triphosphate

19. Hauptseminar Biophysik der Systeme: Chemotaxis19 Phosphoinositide signalling

20. Hauptseminar Biophysik der Systeme: Chemotaxis20 Localization of PIP 3 in the membrane Dicties expressing PH domain of Crac fused to GFP The green crescents align on the edges which are facing the pipette with cAMP  local accumulation of PIP 3 at the leading edge

21. Hauptseminar Biophysik der Systeme: Chemotaxis21 Phosphoinositide signalling PIP 3 - a signalling molecule like cAMP - bound to the cell membrane - activates other signalling molecules (CRAC / PKB) that can bind by Pleckstrin-homology-domain (PH-domain) PH domain PI3 Kinase

22. Hauptseminar Biophysik der Systeme: Chemotaxis22 Phosphoinositide signalling Importance of PIP 3 Dicties without PTEN: - membrane area to which PH Crac -GFP binds increases  amount of PIP 3 increases - extension of the region where actin-filled pseudopodia are extended  PIP 3 enhances actin polymerization and pseudopod formation

23. Hauptseminar Biophysik der Systeme: Chemotaxis23 Imaging of PI3K and PTEN

24. Hauptseminar Biophysik der Systeme: Chemotaxis24 gradient induced localization of PI3K and PTEN when D. discoideum cells are exposed to a gradient, PI3K1 and PI3K2 translocate from the cytosol to the front of the cell, where as PTEN detaches from the anterior membrane and becomes localized to the back of the cell.  mechanism to generate PIP 3 locally at the leading edge this does not dependent on actin cytoskeleton, level of PIP 3, etc.  events controlling the localization of PI3K and PTEN occur upstream of phosphoinositide signalling

25. Hauptseminar Biophysik der Systeme: Chemotaxis25   GG Signalling pathway of dictyostelium discoideum PIP 2 PIP 3 CRAC cAMP PI3K*  PH PTEN Rac/Cdc42 Polarization - Actin polymerization RAS Gradient Sensing pleckstrin homology domain + Acetylcholin- activation

26. Hauptseminar Biophysik der Systeme: Chemotaxis26 Half the Truth!? We discovered: strong link between PIP 3, actin polymerization and pseudopod extension Dicties pi3k1--/ pi3k2– with a chemotactic defect: although directionality and speed of movement is reduced, chemotactic response still occurs!!  In the absence of the PI3K pathway, cells rely on (unknown) underlying mechanisms for gradient sensing!

27. Hauptseminar Biophysik der Systeme: Chemotaxis27 As the time of chemotaxis (i.e. moving of cells) increases, polarity is gradually strengthened. Polarization improves chemotaxis by enhancing the relative sensitivity of the cell at its anterior compared to its posterior.  Ability of response to rapid changes in gradient direction decreases Chemotaxis  Polarization

28. Hauptseminar Biophysik der Systeme: Chemotaxis28 Problem: Hit the Target (…or “Jack, the lonely cell”) 3. Locomotion

29. Hauptseminar Biophysik der Systeme: Chemotaxis29 Polarization: changing the shape by organizing the cytoskeleton Local excitation Global inhibition

30. Hauptseminar Biophysik der Systeme: Chemotaxis30 Organizing the cytoskeleton Actin filaments: - inherent polarity  used to drive membrane protrusions - elastic Brownian-motion mechanism: thermal energy bends the filament and stores energy unbending against the leading edge provides driving force for protrusion

31. Hauptseminar Biophysik der Systeme: Chemotaxis31 Organizing the cytoskeleton Controlling actin polymerization probably mediated by Arp2/3-complex  binds to existing filaments + induces formation of branches Arp2/3 is activated by Wiskott-Aldrich syndrome protein (WASP) + WAVE/SCAR actin polarization is initiated by activation of Rac1 feedback loops help to amplify and maintain Rac activation

32. Hauptseminar Biophysik der Systeme: Chemotaxis32 more intricate network of actin polymerization

33. Hauptseminar Biophysik der Systeme: Chemotaxis33 Organizing the cytoskeleton Myosin-II filaments: - randomly moving Dicty: m-II filaments present in the back + sites of the cell, as well as in retracting pseudopodia - chemotactic moving Dicty: m-II filaments rarely observed in pseudopodia (not retracted) but more are present in the uropod of the cell  chemoattractant gradient regulates the back as well as the front of a moving cell

34. Hauptseminar Biophysik der Systeme: Chemotaxis34 Organizing the cytoskeleton Myosin-II filaments at the back of migrating cells provide the power to retract the uropod. Myosin-II filaments at the sides of these cells suppress the formation of lateral pseudopodia, which otherwise would induce deviations of the cell trajectory. Filaments at both locations support cell polarity.

35. Hauptseminar Biophysik der Systeme: Chemotaxis35 Organizing the cytoskeleton cGMP mediates the formation of myosin filaments in Dicties it is generated by guanylyl cyclases (Sgc/Gca) on chemotactic stimulation counteracting phosphorylation of myosin-II by myosin-heavy-chain kinase: coiled-coil-structure is bended  depolymerizes the filament  coordinating chemotaxis (?)

36. Hauptseminar Biophysik der Systeme: Chemotaxis36 Movement of cells driven by actin Protrusion Adhesion Retraction Three steps of locomotion:

37. Hauptseminar Biophysik der Systeme: Chemotaxis37 Movie

38. Hauptseminar Biophysik der Systeme: Chemotaxis38 Signalling pathway in Dictyostelium

39. Hauptseminar Biophysik der Systeme: Chemotaxis39 Problem: Hit the Target (…or “Jack, the lonely cell”) 1. Directional Sensing 2. Polarization 3. Locomotion

40. Hauptseminar Biophysik der Systeme: Chemotaxis40 Summarization gradient sensing during chemotaxis: complex signalling network - directional sensing PIP 3 -System: sensitizer, ampilfier, director of chemoattractant-induced pseudopod formation (PI3K + PTEN distributed by gradient) - polarization actin polymerization at the leading edge drives protrusion by pseudopod extension cortical myosin filaments at uropod retraction + at the sides suppression of pseudopodia  locomotion

41. Hauptseminar Biophysik der Systeme: Chemotaxis41 Thanks for your attention!

42. Hauptseminar Biophysik der Systeme: Chemotaxis42 References [1] Peter N. Devreotes, Peter J.M. Van Haastert “Chemotaxis:Signalling the way forward”, Nature Reviews 08.2004, Vol.5 [2] Peter N. Devreotes et al., Annu. Rev. Cell Dev. Biol. 2004. 20:22 [3] Peter N. Devreotes et al. Developmental Cell, Vol. 3, 469–478, October, 2002 [4] Bruce Alberts et al., „Molecular Biology of the Cell“ 4th edition 2002 [5]http://en.wikipedia.org/ [6] www.dictybase.org/

43. Hauptseminar Biophysik der Systeme: Chemotaxis43 Eucaryotic Chemotaxis : localized response to chemoattractant Figures : Firtel Gerisch