dynamic processes in cells (a systems approach to biology)

Transcription

1 dynamic processes in cells (a systems approach to biology) jeremy gunawardena department of systems biology harvard medical school lecture october 2015

2 the long road to molecular understanding P&M 1994 Chem 2012 P&M 1971 G-proteins GPCRs cyclic AMP second messengers student P&M 1992 P&M 1947 reversible protein phosphorylation glycogen metabolism student

3 starting at the top X-ray crystal structures suggest an allosteric global toggle switch mechanism Nygaard et al, Trends Pharmacol Sci, 30: ; Rosenkilde et al, Trends Pharmacol Sci, 31:

4 molecular dynamics with purpose-built hardware vestibule 2 adrenergic receptor Dror, Pan, Arlow, Borhani, Maragakis, Shan, Xu, Shaw, Pathway and mechanism of drug binding to G-protein coupled receptors, PNAS, 108:

5 the GTP-GDP switch -adrenergic receptor 1. heterotrimeric G-proteins rhodopsin Gs G G Gt G G transducin 2. small GTPases (RAS, RAN, RHO,... ) OFF ON GEFs GEF = GAP = GDI = GAPs guanine nucleotide exchange factor GTPase activating protein guanine dissociation inhibitor Oldham, Hamm, How do receptors activate G-proteins, Adv Prot Chem, 74: ; Cherfils, Zeghouf, Chronicles of the GTPase switch, Nat Chem Biol, 7:

6 GPCRs are not just coupled to G-proteins... functional selectivity, collateral efficacy, stimulus trafficking,... different ligands can stimulate different patterns of downstream response from the same receptor (perhaps by stabilising different conformations as in allostery) Richard Mailman, GPCR functional selectivity has therapeutic impact, Trends Pharmacol Sci, 28: ; Kenakin, Collateral efficacy in drug discovery: taking advantage of the good (allosteric) nature of 7TM receptors, Trends Pharmacol Sci 28:

7 multi-input, multi-output signalling platform bow-tie structure gives rise to weak linkage ERK -arrestins G-proteins Csete, Doyle, Bow ties, metabolism and disease, Trends Biotechnol 22:

8 Ca2+ is another GPCR second messenger ATP histamine vasopressin carbachol gonadotropin releasing hormone GPCRs G Gq G phospholipase C Ca2+

9 Ca2+ signalling has many second messengers PIP2 plasma membrane DAG 20,000 X cytoplasm variable in different tissues extracellular space ER membrane IP3 endoplasmic reticulum Ca2+ is toxic and at a 20,000X difference in concentration between extra- and intra-cellular compartments Clapham, Calcium signaling, Cell 131: phosphatidylinositol 4,5-bisphosphate PIP2 diacylglycerol DAG inositol 1,4,5-trisphosphate IP3

10 Ca2+ signalling can be very fast neurotransmitter release from synaptic vesicles P&M 2013 vesicle trafficking action potential Ca2+ entry 150 but is highly temperature sensitive Chapman, Synaptotagmin: a Ca2+ sensor that triggers exocytosis, Nat Rev Mol Cell Biol 3: ; Sabatini, Regehr, Timing of neurotransmission at fast synapses in the mammalian brain, Nature 384:

11 Ca2+ signalling toolkit different cell types mix and match components from a toolkit Ca2+ handling/sensitive pumps, channels, receptors, buffers, stores to provide cellular responses appropriate to the cells' physiological roles Berridge, Lipp, Bootman, Nat Rev Mol Cell Biol 1: Berridge, Bootman, Roderick, Nat Rev Mol Cell Biol 4:

12 measuring Ca2+ Aequoria victoria fura2 Ca2+ ratiometric 340/380 excitation 469 nm cameleons aequorin + coelenterazine GFP 509 nm Osamu Shimomura, A short story of aequorin, Biol Bull 2: ; Grynkiewicz, Poenie, Tsien, JBC 260: ; Miyawaki et al, Nature 338:

13 systems and sensors measurements are always a convolution of the system being studied and the measurement sensor measurement system sensor sensor dynamics can dominate the measured response. if the sensor has been characterised, this can be used to deconvolute the response and correct for the sensor's interference but this has rarely been attempted outside of neuroscience However, in fluorescence imaging of Ca transients, the time course of ica is obscured by the presence of the exogenous Ca indicator, which slows the clearance of Ca from the spine... To determine the time course of ica, we corrected for the kinetics of Ca handling by performing a deconvolution with the impulse response of Ca handling of the spine Bloodgood, Giessel, Sabatini, Biphasic synaptic Ca influx arising from compartmentalized electrical signals in dendritic spines, PLoS Biol 7:e

14 = carbachol GnRH = gonadotropin releasing hormone neuron firing pattern hepatocyte CCh gonadotrope = vasopressin insulinoma cells VP hamster egg GPCR signalling parotid gland hepatocyte Ca2+ oscillations Woods, Cuthbertson, Cobbold, Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes, Nature 319: Berridge, Galione, Cytosolic calcium oscillations, FASEB J 2:

15 Ca2+ frequency responsiveness Science 279: Cell 82: EMBO J 15: Nature 392: Cell 95: Nature 392: PNAS 102:

16 different cellular responses changes in signal frequency modulation X oscillations of X with different frequencies how are Ca2+ oscillations generated? how can Ca2+ frequencies be detected?

17 two classes of oscillator architecture receptor-controlled * class II ** IP3 oscillates with Ca2+ 2nd messenger-controlled * class I ** IP3 does not oscillate * Berridge, Galione, FASEB J 2: ; ** Sneyd et al, PNAS 103:

18 positive feedback R = degree of receptor-dependent activation NOT Hill-like functions! K1 is the InsP3 concentration at which half the sites are filled X Meyer, Stryer, Molecular model of receptor stimulated calcium spiking, PNAS 85:

19 the IP3 receptor is a complex beast open statistical factors stay away from them use microstates instead proportion of open receptors X Meyer, Holowka, Stryer, Highly cooperative opening of calcium channels by inositol 1,4,5trisphosphate, Science 240: Foskett, White, Cheung, Mak, Inositol trisphosphate Ca2+ release channels, Physiol Rev 87:

20 positive feedback bifurcation & bistability dy/dt = 0 nullclines R = 0.02 dx/dt = 0 nullcline R = 0.05 R = 0.2 x y Calculations show that cooperativity alone or positive feedback alone do not give rise to bistability. Meyer, Stryer, Molecular model of receptor stimulated calcium spiking, PNAS 85:

21 interlinked negative feedback Ca2+ pump cytosolic Ca2+ Ca2+ in the mitochondria constant leak Meyer, Stryer, Molecular model of receptor stimulated calcium spiking, PNAS 85:

22 3D dynamical system Meyer, Stryer, Molecular model of receptor stimulated calcium spiking, PNAS 85:

23 leads to oscillation increasing signal amplitude signal amplitude to Ca2+ frequency conversion, without affecting Ca2+ peak amplitudes Meyer, Stryer, Molecular model of receptor stimulated calcium spiking, PNAS 85:

24 relaxation oscillators Balthazar van der Pol... was a pioneer in the field of radio... but he also pursued the mathematical problems encountered in radio work so far that his work has formed the basis of much of the modern theory of non-linear oscillations, and he has given his name to the most typical equation of that theory * sharp transition from OFF to ON sharp transition from ON to OFF negative feedback drives hysteresis of an underlying bistable switch slow relaxation followed by fast transition Balthazar van der Pol, On relaxation oscillations, Philosophical Magazine 2: *Mary Cartwright, Balthazar van der Pol, J Lond Math Soc 35:

25 interlinked positive & negative feedback loops neg feedback only neg & pos feedback To construct amplitude vs. frequency curves, we first chose a bifurcation parameter and then identified the range of the parameter over which the system exhibited limit cycle oscillations by locating the Hopf bifurcations at which oscillations were born and extinguished. We then used an iterative algorithm to march along the chosen parameter between the two bifurcations. For each set of parameters we calculated the limit cycle solution of the system, keeping track of the amplitude and frequency of the limit cycle solution. Tsai, Choi, Ma, Pomerning, Tang, Ferrell, Robust, tunable biological oscillations from Interlinked positive and Negative Feedback Loops, Science 321: ; Barkai, Leibler, Circadian clocks limited by noise, Nature 403:267-8, 1999