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CHEMOTAXIS: Signalling the way forward

CHEMOTAXIS: Signalling the way forward. Christian Westermeier. Chemotaxis of Dictyostelium discoideum. Structure of the presentation short introduction signalling pathways: - directional sensing - establishment of polarity - locomotion of the cell summarization. Chemotaxis.

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CHEMOTAXIS: Signalling the way forward

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  1. CHEMOTAXIS:Signalling the way forward Christian Westermeier

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

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

  4. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

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

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

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

  8. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

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

  10. 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) Hauptseminar Biophysik der Systeme: Chemotaxis

  11. 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) Gα2 mainly linked to cAMP-receptors Hauptseminar Biophysik der Systeme: Chemotaxis

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

  13. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

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

  15. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

  16. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

  17. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

  18. Phosphoinositide signalling PIP3 Phosphatidylinositol-3,4,5-triphosphate PIP2 Hauptseminar Biophysik der Systeme: Chemotaxis

  19. Phosphoinositide signalling Hauptseminar Biophysik der Systeme: Chemotaxis

  20. Localization of PIP3 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 PIP3 at the leading edge Hauptseminar Biophysik der Systeme: Chemotaxis

  21. Phosphoinositide signalling PIP3 - 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) PI3 Kinase PH domain Hauptseminar Biophysik der Systeme: Chemotaxis

  22. Phosphoinositide signalling • Importance of PIP3 • Dicties without PTEN: - membrane area to which PHCrac-GFP binds increases • amount of PIP3 increases - extension of the region where actin-filled pseudopodia are extended  PIP3 enhances actin polymerization and pseudopod formation Hauptseminar Biophysik der Systeme: Chemotaxis

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

  24. 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 PIP3 locally at the leading edge this does not dependent on actin cytoskeleton, level of PIP3, etc.  events controlling the localization of PI3K and PTEN occur upstream of phosphoinositide signalling Hauptseminar Biophysik der Systeme: Chemotaxis

  25. g b Ga Signalling pathway of dictyostelium discoideum cAMP + PIP2 PIP3 b g PI3K* PTEN RAS pleckstrin homology domain Rac/Cdc42 Gradient Sensing PH CRAC Polarization - Actin polymerization Acetylcholin- activation Hauptseminar Biophysik der Systeme: Chemotaxis

  26. Half the Truth!? We discovered: strong link between PIP3, 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! Hauptseminar Biophysik der Systeme: Chemotaxis

  27. Chemotaxis  Polarization 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 Hauptseminar Biophysik der Systeme: Chemotaxis

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

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

  30. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

  31. 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 Rac1feedback loops help to amplify and maintain Rac activation Hauptseminar Biophysik der Systeme: Chemotaxis

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

  33. 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 Hauptseminar Biophysik der Systeme: Chemotaxis

  34. 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. Hauptseminar Biophysik der Systeme: Chemotaxis

  35. 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 (?) Hauptseminar Biophysik der Systeme: Chemotaxis

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

  37. Movie Hauptseminar Biophysik der Systeme: Chemotaxis

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

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

  40. Summarization • gradient sensing during chemotaxis: complex signalling network • directional sensing • PIP3-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 Hauptseminar Biophysik der Systeme: Chemotaxis

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

  42. 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/ Hauptseminar Biophysik der Systeme: Chemotaxis

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

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