Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Endomembrane system wikipedia , lookup
Tissue engineering wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell growth wikipedia , lookup
Cytokinesis wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cell culture wikipedia , lookup
Cellular differentiation wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Signal transduction wikipedia , lookup
The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 Chemotaxis Cells developed the capacity to move in order to feed and to avoid local harmful situations. They are attracted to all sorts of stimuli and are repulsed by others. The term "Chemotaxis" was first coined by a W. Pfeffer in 1884 to describe the attraction of fern sperm to the ova, but since then the phenomenon has been described in bacteria and many eukaryotic cells in many different situations. Specialised cells within metazoans have retained the ability to crawl toward bacteria in order to eliminate them from the body and the machinery is very similar to that used by primitive eukaryotes to find bacteria for food. Much of what we know about chemotaxis has been learned from studying the slime mould Dictyostelium discoideum, and comparing this to our own neutrophils, the white blood cells that detect and consume invading bacteria in our bodies. Neutrophils are end differentiated and largely non-biosynthetic cells which means that we cannot use the usual molecular biological tools that we would other wise use, such as gene knockout, transient transfection, or expression of GFP-labelled proteins. Fortunately, Dictyostelium can be used for all such studies. An understanding of neutrophils chemotaxis is of obvious importance for the treatment of human disease and therapeutic intervention of these processes has resulted. Whereas chemotaxis is the sensing of a chemoattractant gradient and climbing it cells can also accumulate by chemokinesis. Chemokinesis is an activity that increases the overall speed of locomotion. In principle a cell be perceive gradients by a number of distinct mechanisms (Figure 14). These hypothesis have been offered in various guises by a number of authors. t2 C Temporal t2 t1 mC e Spatial h R2 R1 P1 oh P2 t o h tm C ae Pseudospatial R2 R1 ho a t Cm r a e Temporo-spatial Figure 14. How cells may perceive chemo attractant gradients (after Lackie, 1986). The temporal model assumes that the amoeba measures and "remembers" the chemo attractant concentration at time zero and compares this to a second reading at some time point later as the cell crawls. In this way the amoeba gets clues like that particularly annoying party game "getting warmer" / " getting colder" (Arghhh!!). The spatial model assumes that the cell is able to read the concentration across the span of the cell so that it can compare the number of chemoattractant molecules. A related model is the pseudospatial model, where the cell tests concentrations by sending out pseudopods (P1 and P2 ) at various points to sense where the highest concentrations lie. The temporo-spatial model, detects waves of chemo -attractants coming toward the cell from a particular direction. Note that this last mechanism could not read a static gradient. A huge amount of work has been expended trying to work out which (if any) of these hypotheses is correct. to a a mt t r ea c t The distribution of cAMP receptors on Dictyostelium has been determined by replacing the gene with a gene consisting of the receptor fused to green fluorescent protein (GFP –see below). These studies (Xiao et al, 1997) indicate that there is no particular concentration of receptors within pseudopods as expected from the spatial, pseudospatial or temporospatial models. However we cannot discount these models on this basis since they could all function in principle without concentrated receptors (it would just work better). The temporo-spatial model cannot be valid for all cases as it is well established that cells can chemotax in stable gradients of attractions. tt 7 nc aa t tr n ct a at t t ra n c The Boyden chamber. Two chambers are separated by a filter through which cells migrate (page 7, figure15). Chemotactic gradients can be set up by placing different concentrations of the putative chemo -attractant in the upper and lower chambers. The advantage of the Boyden chamber is that is can discriminate between chemo -kinetic and chemotactic influences. The use of this chamber requires that the cells under test have to move in three dimensions (most do) and to squeeze between the pore size of the particular filter. Filters are available in different average pore sizes, so this latter point is seldom a problem. The Boyden chamber is reproducible and the chemokinetic, chemotactic response easy to quantify. The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 Upper chamber Top Bottom Filter Lower chamber Media 1/1000 1/100 1/10 Media 0.2 0.2 0.3 0.5 1/1000 0.8 0.6 0.4 0.4 1/100 3.4 3.5 0.7 0.6 1/10 2.7 2.3 0.8 0.2 % of Cells in Bottom Chamber Figure 15. The Boyden Chamber. The main advantage is that it can detect chemokinetic effects as well as chemotactic effects. The Checkerboard assay (right) is a way to organise the Boyden chamber experiment. .8 Figure 16. The checkerboard assay can be used to differential chemokinesis from chemokinesis. If attractants are added to both chambers at various concentrations, the relationship of migration to attractant can be plotted. In this example, the attractant is both a chemokinetic and a chemoattractant. .6 .4 .2 2 0 0 0.05 0.1 0.15 Dilution of Chemo-attractant Control Attractant Attractant Cells Control Cells Figure 17. The under agarose method (left) versus the over agarose method. The under agarose method (Laevsky & Knecht, 2001 ) is simple and can be used for a variety of cell types. Chemo -attractant gradients are set up as the attractant diffuses from the trough into the agarose. The presence of the agarose stabilizes the gradient that might otherwise be dispersed or changed by thermal motion or minor agitations. An advantage of the various under agarose methods is that it is possible to set up multiple gradients of various chemo -attractants to study their effect on cell behaviour simultaneously (Heit et al, 2002). Cell chamber Figure 18. A stable gradient can be created by placing the putative attractant in one well and the cells are then placed in a second well. Chemo-attractant chamber Agarose Cells Petri dish 8 The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 Chemotaxis in Development Mammalian development begins with the meeting and fusion of the gametes, the female egg gives signals and the male sperm comes swimming (setting the pattern for life?). Chemotaxis helps the sperm find the egg in humans (Eisenbach & Tur-Kaspa, 1994), and in algae where a chemo -attractant increases the turning angle of the sperm cell so that it spirals in toward the egg like a captured moon. Development involves mass movements of cells. Attractant fMLP C5a IL-8 LTP4 Type End target End target intermediate intermediate Transduction pathway p38 MAPK & PI(3) Kinase p38 MAPK & PI(3) Kinase PI(3) Kinase PI(3) Kinase Function Liberated by bacteria, allows neutrophils to follow them. Produced from inflammatory lesions via complement Liberated from other cells to attract neutrophils Liberated from other cells to attract neutrophils Table 1 Chemoatrractants for Neutrophils Figure 19. Neutrophil migration into inflammatory regions. (1). Neutrophils in the microvascular vessels circulate passively in the blood. At sites close to inflammation (2), the endothelial cells alter their charge in response to interleukins such as IL-8, so that now neutrophils can stick. (3). Neutrophils now crawl along the "-dimensional surface of the vessel and then squeeze between the endothelial cells. (4) Neutrophils can now invade the 3-dimensional space and begin to detect the fMLP and/or C5a gradient at the same time as a negative IL-8, LTP4 gradient. It has been suggested (Heit et al, 2002), that such an arrangement is more stimulatory. (5) Neutrophils arrive at the site of infection drawn by the gradient and now consume the bacteria by phagocytosis. Eventually the neutrophils die full of now dead bacteria and accumulate as pus. The top line in the graph represents adhesive forces which peak fMLP IL-8 LTP4 C5a Adhesion Chemoattractant at the endothelial surface, drop after that as the neutrophils crawl in the 3-dimension matrix. Adhesion then increases with fMLP/C5a gradient. The lower graph represents the expected gradient of the various classes of chemo -attractants. Transduction of Chemo-attractant Signals Most of what is known about the signalling cascade has been gleaned from Dictyostelium, but recent contributions are available from knock out mouse models. From the top, chemotaxis requires the chemotactic receptor, heterotrimeric G- Ligand GPCR γ γ Signal P L C β Gα GTP PIP2 PI(3)K IP3 PIP2 PI(3,4,5)P3 + DAG Superoxide production Ca2+ PKC Activation of cytoskeleton for chemotaxis 9 Figure 20. G-protein linked chemo attractant receptor dissociates trimeric G-protein and the b,g subunit activates both PLC and PI(3)-kinase. The products of PI(3)kinase are short-lived messengers that bind a number of targets leading to their activation. The activation of PLC is important for activation of the superoxide burst after phagocytosis in neutrophils but not important in chemotaxis its self. Mice bred without PI(3)-kinase however have a reduced capacity to chemotax. The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 proteins, Whereas the distribution of chemotactic receptors is uniform across the cell (Xiao et al, 1997), other signalling molecules further down the cascade are found to have a polarized distribution. Mammalian PLCγ become localised to the leading edge in a PI3-Kinase dependent manner (Piccola et al, 2002). PLCγ contains a PH domain that binds PtdIns-3,4,5-P3, a product of PI3-Kinase, and so its probable that the PLCγ localization is downstream of PI3-Kinase activity. The βγ subunit of the activated hetero-trimeric G-proteins are weakly polarised fashion with concentrations at the leading edge (Jin et al, 2000). Another protein involved in chemotactic pathway AKT (protein kinase B), also contain PH domains and are also localized to the leading edge of cells (neutrophils)(Servant et al, 2000). Surprisingly, it has recently been reported that in mouse neutrophils, PLCs are not required for chemotaxis but are involved in priming the superoxide burst (Li et al, 2000). Dictyostelium too seems to chemotax in the absence of PLC (Drayer et al, 1994). The role of PI (3) kinase and p38/MAPK in chemotaxis A number of studies have demonstrated the importance of the PI3-kinases in chemotaxis of both neutrophils and Dictyostelium (and a number of other cell types). Most convincing are experiments where the genes for these enzymes have been deleted (Hirsch et al, 2000). These experiments indicate that in the absence of PI3-kinase most cells do not RAC P21-activated kinase PAK1 LIM Kinase CDC42 PI(3)K P RAS p21 G-proteins PIP2 PI(3,4,5)P3 p38 MK2 Hsp27 MK2 Hsp27 AKT PKB Figure 21. The p21-activated kinase (PAK1) is as its name suggests normally activated by members of the p21 G-protein family, CDC42 and Rac. In addition to this route it has been found that activated PI(3)kinase also activates PAK1 even in the absence of either p21 (Papakonstanti & Stournaras, 2002). PAK1 activates LIMK by phosphorylation Activated enzymes are indicated with a flash symbol. chemotact, however some cells still did. It is suspected (Heit et al, 2002) that the remain cells respond to chemo attractants through the p38-MAPK pathway. Full activation of chemotaxis involves the phoshorylation of AFT/PKB and although this is partially achieved through PI3-kinase is seems that additional phosphorylation by p38 is require at a distinct site. Name ENTH FYVE PH PX Derivation Epsin N-terminal Homology Binds to PI (4,5) P2 & PI (3,4,5)P3 Proteins containing Epsin, Fab1p, YotB,Vac1p, EEA1 Pleckstrin Homology PI (3)P PI 4,5 P2 Early endosome antigen PLC, Unc104 Kinesin, Spectrin Phox Homology PI (3)P Table 2 Protein domains that bind phosphatidylinositol lipids Receptor PI(3)K PAK PI3P PTEN Chemo-attractant gradient 10 phox p40 Figure 22 The distribution of the molecules primarily involved in chemotactic signalling. The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 Human diseases resulting from failed chemotaxis Chemotaxis is such an important defence mechanism that it is not a surprise that pathogens have developed toxins that inhibit the process. These toxins are of course bad news for the infected, but they have provided researchers with a valuable set of tools for the study of chemo taxis. Staphylococcus aureus inhibits neutrophil chemotaxis by producing CHIP a small 14kDa protein that binds to and inactivates the receptor for FMLP and C5a. Bordetella pertussis is a gram bacteria that is responsible for whooping cough. It produces a toxin that ADP-ribosylates the G-protein that transduces signals from the receptor and many bacteria e.g. Clostridium produce toxins that target the actin cytoskeleton so that the cells can no longer crawl. Staphylococcus aureus Bordetella pertussis Clostridium difficile Clostridium botulinum CHIP Pertussis toxin Toxin B C3 toxin These toxins can be used as very specific tools for research. For example Clostridium toxin B, can ADP-ribosylate and so inactivate Rho type monomeric Gproteins thereby inactivating them. Neutrophils can be loaded up with PI3P lipids if histones (basic proteins) are used as carriers (Weiner et al, 2002). When the cells are loaded up they spontaneously polarise and more PI3P is produced in a positive feed back mechanism. The absence of Rho proteins (through toxin B treatment) prevents this occurring so that the feedback mechanism involves Rho proteins. The details of this important mechanism are not yet certain. Receptor G-protein Rho G-protein Actin Ligand Activation by Ligand binding Staphylococcus aureus toxin γ γ Gα GDP γ β β Gα β GTP Bordetella pertussis toxin Signal Figure 23 Many others target the cytoskeleton Ex1 Em1 Ligand Activation by Ligand binding Ex2 Em2 γ Gα γ β CFP EFP Figure 24 γ β Gα EFP β CFP Wave length Emitted yellow light Another use for green fluorescence protein (GFP) technology allows us to monitor the interactions of specific proteins in living cells by using fluorescent energy transfer (FRET) combined with digital imaging. Two variants of the standard GFP are made by the introduction of mutations around the site involved in the fluorophore so that GFP now emits at cyan (cyan fluorescence protein) while another set of mutations changes the absorption spectra of another protein (yellow fluorescence protein) matches the emission of the CFP. When the molecules (CFP and YFP) are close light emitted by CFP is absorbed by the YFP which then emits this at a longer wavelength (yellow-green range). If as in the case above, the CFP is fused with Gα, while YFP is fused with Gβ subunit, then the state of the complex can be monitored by fluorescence microscopy. When cAMP is added to the Dictyostelium transfected with the two constructs, a reduction in fluorescence is measured indicating that the complex has dissociated. This experiment now tells us 11 The Dynamic Cell Chemotaxis S.K. Maciver Jan '03 where the G-protein is activated, and the kinetics of the dissociation. Thus information about the complex can be measured in living cells – completely unthinkable even a few years back!! The GFP revolution only really started in 1990(ish) when it was introduced to cell biology. GFP forms also promise to reveal intracellular pH transients and even molecular clocks as there are forms (mutants) of the protein that age within the time scale of hours changing their fluorescent spectra!( Terskikh et al, 2000). The take home message of all of the above is that the cell signalling molecules are arranged at the plasma-membrane by the phosphatidylinositol lipids especially the more phosphorylated forms such as PIP2 and PI3Ps. In the next lecture we will see that these lipid messengers also organise the cell body by the effects of the cytoskeleton. Myosins are crucially stimulated to form multi-meric complexes and further stimulated to contract. References:Drayer, A. L., Van der Kaay, J., Mayr, G. W. & Van Haastert, P. J. M. (1994) Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity. EMBO J. 13, 1601-1609. Eisenbach, M. and Tur-Kaspa, I. (1994) Human sperm chemotaxis is not enigmatic anymore. Fertil. Steril. 62: 233-235. Funamoto, S., Meili, R., Lee, S., Parry, L. & Firtel, R. A. (2002) Spatial and temporal regulation of 3-phosphoinositides by PI 3kinase and PTEN mediates chemotaxis. Cell. 109, 611-623. Heit, B., Tavener, S., Raharjo, E. & Kubes, P. (2002) An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J. Cell Biol. 159, 91-102. Jin, T., Zhang, N., Long, Y., Parent, C. A. & Devreotes, P. N. (2000) Localization of the G protein βγ complex in living cells during chemotaxis. Science. 287, 1034-1036. Lackie, J. M. (1986) Cell Movement and Cell Behaviour, 1 eds, Allen & Unwin, London, Boston, Sidney. Laevsky, G. & Knecht, D. A. (2001) Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and mechanically inhibited conditions. BioTechniques. 31, 1140-1149. Papakonstanti, E. A. & Stournaras, C. (2002) Association of PI-3 Kinase with PAK1 Leads to Actin Phosphorylation and Cytoskeletal Reorganization. Mol. Biol. Cell. 13, 2946-2962. Piccolo, E., Innominato, P. F., Mariggio, M. A., Maffucci, T., Iacobelli, S. & Falasca, M. (2002) The mechanism involved in the regulation of phospholipase Cγ1 activity in cell migration., Oncogene. 21, 6520-6529. Pollack, E. D. and Muhlach, W. L. 1981. Stage-dependency in eliciting target-dependent enhanced neurite outgrowth from spinal cord explants in vitro. Dev. Biol. 86: 259 - 263. Niggli, V. & Keller, H. (1997) The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptideinduced locomotion and increases in cytoskeletal actin in human neutrophils. Eur. J. Pharmacol. 335, 43-52. Ralt, D., Manor, M., Cohen-Dayag, A., Tur-Kaspa, I., Ben-Shlomo, I., Makler, A., Yuli, I., Dor, J., Blumberg, S., Mashiach, S. and Eisenbach, M. (1994) Chemotaxis and chemokinesis of human spermatozoa to follicular factors. Biol. Reprod. 50: 774-785. Rane, M. J., Coxon, P. Y., Powell, D. W., Webster, R., Klein, J. B., Pierce, W., Ping, P. & McLeish, K. R. (2001) p38 kinasedependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J.Biol.Chem. 276, 3517-3523. Servant, G., Weiner, O. D., Hertzmark, P., Balla, T., Sedat, J. W. & Bourne, H. R. (2000) Polarization of chemo attractant receptor signalling during neutrophil chemotaxis. Science. 287, 1037-1040. Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I. & Siebert, P. (2000) "Fluorescent timer": Protein that changes color with time., Science. 290, 1585-1588. Wang, F., Herzmark, P., Weiner, O. D., Srinivasan, S., Servant, G. & Bourne, H. R. (2002) Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils. Nature Cell Biol. 4, 513-518. Weiner, O. D., Neilsen, P. O., Prestwich, G. D., Kirschner, M. W., Cantley, L. C. & Bourne, H. R. (2002) A PtdInsP3- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity. Nature Cell Biol. 4 , 509-512. Xiao, Z., Zhang, N., Murphy, D. B. & Devreotes, P. N. (1997) Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation. J. Cell Biol. 139, 365-374. http://dictybase.org/tutorial/spatial_gradients.htm Queries to S. Maciver Room 444, Hugh Robson Building, George Square. E-mail [email protected]. Fax/Tel 0131 650 3714. 12