Intercellular signalling Flashcards
(23 cards)
Plants: reasons to control photoperiodism
Coupling oscillators can create a dynamic system that feeds back into itself to create complex behaviours. Dev switch from vegetative to flowering growth reg cell fate. (NB: skotoperiod= dark period of day)
Reasons to control flowering time: cross pollination (flower together with others in same species); pollinator availability- insects active in warm weather, favour spring/summer flowers-> co-evolution of plants w/ pollinators results in temp+ photoperiod being important flowering cues; resource availability- large flowers appear later when more Photosynth has occurred, producing sufficient nutrients for flower dev; also need sufficient carbon store for sufficient fruit development.
Impact of flower timing and agricultural significance
Impact of flower timing: growth range offers opportunity for intervention (alter flowering time) for max yield, e.g. double cropping (2 crops in 1 season). Also select for diff flowering times to extend agricultural flexibility + eco-geographical range of crops.
Agricultural significance: Cereals derived from wild relatives in Fertile Crescent that flower in response to long days after vernalisation, ensuring grain filling happens before dry+ hot summer. Agriculture spread to Europe req selection of adaptive traits for new env- early farmers 8500-5000yrs ago likely picked photoperiod-insensitive mutants to grow in long+ moist European summer. Alleles of Ppd-H1 gene partially ctrl barley flowering time, have been cloned to ID point mutations in history. Wild type Ppd-H1 from SW Asia, S Eur+ Mediterranean basin-> early flowering in long days (Winter barley- grows in winter, flowers in spring). Central+ N Eur barley has photoperiodic-insensitive ppd-H1- spring barley- grows in spring, flowers in late summer. Ppd-H1 = orthologue of Arabidopsis AtPRR7 involved in circadian clock function.
Photoperiodism and coordinating sensing response
Photoperiodism: ctrl reproductive behaviours in many animals and plants. Seasonal changes measured-> reproduce at optimal time. Long day plants (e.g. Arabidopsis- flowers much slower (61 days after) if planted in winter solstice compared to summer (29 days) and is much more developed @ flowering time (42 vs 10 leaves)) flower when photoperiod> critical value. Short day plants vice versa. Some plants photoperiod insensitive.
Coordinating sensing+ response: in postembryonic plants, dev stage changes occur in apical meristem (AM) (alters dev programme of plant- site of response, progress though juvenile, adult vegetative+ reproductive phases). Leaves sense photoperiod- info must be transduced to AM to produce appropriate response. Intracellular signalling during photoperception in leaves, then intercellular from leaves-> AM+ intracellular again for dev fate change in AM cells.
Arabidopsis circadian clock
Arabidopsis’ circadian clock comprises oscillator of inter-reg repressive TFs- LHY, CCA1, TOC1. LHY+CCA1 are MYB-like, activated at dawn by phytochromes+ cryptochrome (red+ blue receptors), bind TOC1 promoter evening element, repress exp. CCA1/LHY break down in PM, TOC1 exp, represses CCA1/LHY exp. TOC1 broken down just before dawn, resetting cycle.
Plants: competence
Competence: in early dev, AM produces vegetative tissue regardless of env. Later, dev competence to respond to intercellular photoperiodic signals from leaf; then, becomes determined to flower, even if plant returns to non-inducing photoperiod. Arabidopsis competence changes due to daylength-independent decline in micro-RNA miR156 hairpin transcribed by POLII+ generating 21-nt siRNA via DICER. miR156 targets= fam of TFs- squamosa promoter binding like (SPLs)- regulating flower promoting gene exp. miR156 (+ other miRNAs) promote juvenile phase, decrease w/ increasing sugar in plant (incl tetrahalose-6-P) as it matures (age sensor).
Plants: coincidence model overview
Coincidence model: Bunning, 1933- proposes circadian clock drives 24hr rhythms in light-sensitive regulator of flowering -clock-regulated protei-> production/ accum./ regulator only @ certain time of day; flowering induced by coincidence in the regulator+ light presence, i.e. light can only cause flowering if present at a certain time of day. (external cue (light) coincides with internal cue (regulator, abundance regulated by circadian clock) in long days to produce response by flowering gene exp- seen in Arabidopsis)
Coincidence detector= CONSTANS (CO) found in mutant screens- a B-box type Zinc Finger protein (Transcriptional activator). Constans mutants flower late in long days but @ same time as wt in short days. CO mRNA abundance oscillates under constant light-> exp ctrl by circadian clock. Note that constans exp only in leaves+ phloem, not ap. Meristem.
Plants: coincidence model step-by-step
Activation by coincidence:
1) CO mRNA under circadian ctrl by strling CYCLIN DOF FACTOR1 (CDF1) abundance- TF w/ 1 Zn finger motif that represses CO transcription when bound to its promoter.
2) CDF1 degraded by SCF E3 UQ ligase in afternoon, allowing CO transcription.
3) CDF1 degradation only in PM as F-box protein FKF1 (part of SCF) transcribed only then.
4) Morning- FKF1 transcription blocked by MYB-like TF CCA1 binding ‘evening element’ in FKF1 promoter. CCA1 transcript abundant in morning due to light activation of promoter+ because CCA1 part of circadian clock. In PM, CCA targeted for degrad by another E3 UQ ligase.
5) COP1 E3 UQ ligase-> CO protein degrad in dark. Light-> conf change in crypt+ phytochromes, allowing them to interact+ reg target proteins; CRY binds COP1 inhibits cullin activity/ COP1.
6) If Light+ CO mRNA peaks coincide, CO translated+ accumulated.
7) CO protein accum-> flowering locus T (FT) exp+ flowering. CCA1 also reg FT exp by CO-independent pathway.
Mathematical model for coincidence in Arabidopsis; light sensing in plants
Can construct mathematical model for Arabidopsis FKF-CO-FT genetic network+ its reg by clock+ light (1st math model for photoperiodism), allowing formal testing of coincidence model. Simulations using the model may aid engineering designer crops w/ flowering fit for local env.
Aside on light sensing: Cryptochromes also found in insects, contains FAD as chromophore reduced by light-> conf change; mutants in cryptochrome produce flowering time phenotype. (CRY-COP-DET1 also found in mammals for targeting reg proteins, but better understood in plants). In phytochrome, Pr->Pfr by red light an back by far red; Pr ½ life ~30 min, Pfr ~4.5 hrs. Pfr activated cellular processes. Phytochromes form dimers as platforms for protein-protein interactions
Florigen
Florigen= long distance signal for flowering. Chailakhyan, ‘30s proposed its existence+ that is travel in phloem from leaves-> AM, promote flowering. Existence demo’d by grafting leaves from plants grown in flowering photoperiods on to stock grown in non-flowering env-> leaves cause flowering. Florigen signal is self-propagating- once flowering induced by grafting, leaves in non-inductive stock become competent to induce flowering in non-inductive stock too- shows florigen transmits via phloem, causes further florigen production. Recent finding that circadian clocks located in vasculature cells contributing to reg/flowering time supports this. Florigen= FT locus protein, thought to encode RAF kinase inhibitor-type protein. FT:GFP fusions found in leaves+ phloem. FT (protein, not mRNA, as previously though) exp (not CO) in AM-> flowering- hence it is the long-distance signal between leaves+ AM.
Flowering locus C and vernalisation; epigenetic reprograming
FLC: Flowering locus C represses FT exp, regulates competence to respond to photoperiodic signals. FLC= MADS box TF, exp induced by FRIGIDA, regulates competence to respond to photoperiod signals, integrates vernalisation+ autonomous pathways into flowering response.
Vernalization: cold supresses FLC exp via vernalisation gene (VRNs+ VINs)-dependent methylation/ H3.
1) H3 VIN3 induced by cold, req for chromatin state that inactivates FLC.
2) VIN3-> histone deacetylase (direct or indirect- uncertain)- exp induced after 30-40 days of cold. Histone deacetylation+ H3 K27 methylation-> FLC heterochromatin.
3) VIN2= polycomb for maintenance of heterochromatin+ inactivation/ FLC for the rest of the plant’s life (homologue/ Droso polycomb group gene Sn(Z)12 thought to maintain H methylation). Repression allows FT/ flowering gene exp.
Epigenetic reprogramming allows FLC exp in next gen- ‘memory’ loci reset each gen, otherwise epigenetic marks accum, reg mech blocked. FLC H3 K27 Me3 reversed in gametes+ during embryo dev by EARLY FLOWERING 6- H3K27me3 demethylase, allowing FLC exp to stop seedlings flowering before vernalisation.
Polycomb proteins, autonomous pathway and arabidopsis as a model organism
Polycomb proteins recruited to FLC by binding COLDAIR (non-coding RNA). COLDAIR promoter+ sense seq are in 1st intron/FLC, exp induced by cold. H3 Me+ other steps-> heterochromatin @FLC. Methylation not reversed by warm weather, so FLC abolished until next gen. Unclear if coldair recruits Su(z)12 polycomb in addition to E(z) class, Antisense transcripts/ COOLAIR may also silence FLC.
Autonomous pathway: causes eventual flowering regardless of conditions (old age response). Epigen mods incl chromatin remodelling+ RNAi repress FLC. Cellular miR156 drops.
Arabidopsis as a model organism: easy to grow; fast life cycle; sexual reproduction AND self-fertilisation; easily transformed/ mutated; seq available+ established; orthologues of many genes have flowering reg roles in cereals.
Animals: insulin signalling and production
Production in pancreatic islets of Langerhans glucagon production in alpha cells, insulin in beta.
Diabetes mellitus est. affect 537mln people, caused by hyperglycaemia. Type I= loss/ beta cells (autoimmune response). Type II= alterations to insulin action (reduced sensitivity- mech unclear)
Insulin production: polpypep hormone, initially made as preproinsulin. Signal sew directs it to ER, where removed-> proinsulin-> Golgi+ sec vesicles, where 33aas removed from middle-> insulin (held together by S-S, stored in vesicled for release). Purified by Banting+ Best, seq by Sanger, structure by Hodgkin. Rise in blood glucose/hormones-> increase insulin release- conc in blood v low (order of 10-10M)
Insulin level regulation by glucose
Glucose. Mech studied in isolated islets in immortal mouse beta-cell line MIN6. Glucose transported by passive GluTs (fam of 14, 1-4 well characterised). Mouse beta cell glucose flux via GluT2 (GluT1 in humans), constitutive. Glucose->G6P by GK to maintain inward diff gradient. G6P eventually-> ATP synth. Adding GK inhibitor mannoheptulose stops glucose stimulated insulin release- conversion to G6P req. Studies using patch clamp techniques show that [ATP]= signal for insulin release.
Insulin and secretion-stimulus coupling
Secretion-stimulus coupling. Studies using electrophysiological techniques (as above)+ molecular bio elucidate mech. beta-cell mem is polarised by ion channels/pumps ~-70mV. Status quo esp maintained by K+ efflux via KATP channel (target for sulphonylurea drugs for type II diabetes, which trigger insulin release by acting on SUR subunit, inducing K channel closure)- rise in [ATP] closes channel, K+ efflux drops, mem depolarises. Voltage dependent Ca channels (L-type) reg by me potential- as it reaches threshold potential, channels open-> Ca rapid efflux-> action potential observable by electrophysiological approaches. [Ca] rise-> insulin loaded sec vesicle fuses mem- stimulus-secretion coupling (analogous to excitation-contraction coupling in muscles, also Ca dependent. Some similarity to neurotransmitter release).
Insulin signal termination and oscillating Ca signals
Signal stop: return of excitable beta cells to resting state involves 2 mechs:
1) SK channels- K+ channels that open in response to rise in [Ca], allowing K+ efflux.
2) 2nd mech part of generic Ca signalling system. Cytoplasmic [Ca] tightly ctrled. Xs quickly removed by pumping out of cell (plasma me Ca ATPase, PMCA) and into intracellular stores (SERCA) actively, reducing [ATP], increasing K-ATP channel opening, reducing [Ca]. Channels diff, but voltage-dependent ion influx followed by feedback mech analogous to other action potentials (result= mem pot oscillation)
Oscillating Ca signals: [Ca] observed using Ca sensitive dyes (fura-2, fluo-4). In constant [glucose], ca signal oscillates- high freq oscillations correspond to action pots superimposed on lower freq pattern (period 4-6 min- various models proposed). Glycolytic activity+ [ATP] may oscillate w/ it @ this lower freq, oscillators interacting-> complex pattern.
Cell coordination in an islet of Langerhans
Cell+ Islet coordination: individual islet cells’ [Ca] and insulin release oscillate via unstable system w/ feedback loops, coordinating action within islet. [insulin] in blood exiting pancreas also oscillate w/ same 3-6 min period- islets within pancreas also coordinate. Intact islet imaging shows Ca signal coordination @ higher time res than 4-6 min periods.
Cell coordination in an islet: Rocheleau used transgenic mouse model exp K-ATP channel w/ inactive ion channel tagged w/ GFP. When mutant K-ATP channel exp in cells containing wt channel, it acts as dominant-negative as wt channel= tetramer, incl even 1 mutant subunit inactivates. When transgene introduced in mice, they had mild hyperinsulinism, w/ only 70% islet cells exp transgene (+ non-functional K-ATP). Insulin release measured by radio immune assay. Intact islets containing transgene have synched Ca2+ signals – so even cells w/ out f(x) KATP have normal Ca2+ signalling, glucose-dep insulin release. If cells dissociates, transgene cells don’t show normal Ca signalling/ glu-dep insulin release- so 30% normal cells confer wt behaviour to islet- suggests cells electrically coupled so individual oscillators coupled, confirmed by Ca signal velocity. Most likely coupling mech via gap junctions- adding gap junction inhibitor (18 alpha-glycyrrhetinic acid) mimics effect of dispersion (cells behave as individuals), supporting this; ablating gap junction protein connexin 36-> loss of synchronisation. Beta cells behave as e- syncytium-> able to release insulin pulses. NB gap junctions =/= route for beta cell comms.
Islet coordination and regulation by neurotransmitters
Islet coordination in a pancreas: mech unclear, islets appear to drive oscillations. An isolated pancreas oscillating as whole doesn’t necessarily req external input. Suggested that ACh or ATP may act to sync islets. Regulation by neurotransmitters: ACh released before+ during feeding. Use G-protein coupled receptor+ 2nd messenger mediated cascades. G-protein coupled receptors have 7 TMDs, external NTD, internal CTD, couple heterotrimeric G-proteins made of alpha, beta+ gamma subunits. Alpha binds GD/TP, active when GTP. Beta+ gamma act as single subunit. Diff types/alpha reg diff 2nd messenger systems. Galphaq couples phospholipase Cbeta (PLC) activation, I and s-> cAMP. PLC cleaves mem lipid PI-4,5,P2-> DAG (lipid moiety pf PIP2) which stays in mem+ IP3 (soluble head group. IP3 receptor= large tetrameric protein in ER. IP3-> IP3R opening, Ca release into cytoplasm-> insulin release. Signal terminated by pumps (as above). One suggested model/ islet coordination based on mathematical modelling: intrapancreatic ACh initiates intracellular Ca signals, which sync Ca oscillators- still need experimental testing.
Purpose of oscillating signals and human islets of Langerhans
Oscillation purpose: possibly stop receiving cell desensitisation by constant high insulin, esp important in liver. Importance oscillation indicated by: Type 2 diabetes often assoc. w/ disrupted synchronisation; pulsatile application/ exogenous insulin more effective at ctrling blood glucose that continuous.
Human islets: most exp work on mouse islets. Human islets harder to come by+ work with, appear to have more complex architecture so beta-cells appear less interconnected. Results mixed; recent data suggest human islets oscillations coordinated+ insulin release from pancreas pulsatile, w/ evidence of oscillatory behaviour disruption in type 2 diabetes. Overall, coupled oscillators-> cells acting in phase/ more complex ways; signal syncing may coordinate islets.
Insulin mode of action and the insulin receptor
Initially believed to act on liver, now know action on muscle (substantial gluc sink), adipocytes also v important.
Muscles+ adipocytes have low glucose uptake @ resting [insulin]. Glucose uptake passive via GluTs, major GluT in insulin sensitive tissues=4- not constitutively present in membrane. Addition/ insulin to adipocytes-> 10-20 fold increase in glucose uptake in 15 min- too fast for de novo GluT4 synth. Can fuse GFP-> GluT4, intro transgene into cells to visualise. When no insulin, GluT4 stored in vesicles. Add insulin-> rapidly goes to mem.
Insulin receptor= RTK. Insulin affinity ~10-10M. has 2 alpha+ 2 beta chains (produced by cleavage of single polypep)- dimer. Alpha extracellular, insulin binding site. Beta cross mem, have Tyr kinase domain. Insulin binds-> receptor conf change-> auto-pi- stabilises active state+ pi-Tyr provides docking sites for other proteins. Insulin receptor substrate (a docking protein, fam of 4 proteins in mammals) binds. IRS has PTB domain to bind PiTyr+ PH domain- binds in plasma mem. Multiple IRS residues Pi’d. Pi-IRS-1 becomes scaffold for signalling proteins. PI3 kinase binds (part of PIP3 signalling system)
PIP3 signalling, PDK
PIP3 signalling PI3K: PIP2 Pi’d again by class 1 PI3K-> PIP3. PI3K activated by tyr kinases or some GCPRs, has catalytic+ reg subunits. Reg subunit of class 1 PI3K has SH2 domain to bind PiTyr on IRS+ other activating proteins, hence recruited to plasma mem to make PIP3 close to activated receptor. Exps where Pi3K activity dropped (e.g. by inhibitor wortmannin)/ increased show PI3K important to GluT4 translocation+ that more pathways exist downstream.
PIP3=2nd messenger (produces in response to signal, broken down by dedicated ‘off’ mechs- in this case phosphatases SHIP+ PTEN)- increase transient, has specific effector proteins. PIP3 confined to plasma mem, can’t diffuse to cytoplasm. Recruits proteins to PM, in many cases via PH domains (100-120aas, primarily bind PIPs, localising protein to mem). Proteins w/ PHs binding PIP3 go to PM when PIP3 made, observable by GFP labelling PH. In insulin signalling, 2 Ser/Thr kinases PDK1+ Akt1 (AKA PKB) recruited via their PHs.
PDK1 constitutively active bc constitutively auto-pi’s. PIP3 recruits it to mem, bringing it to its target Akt. Akt Pi sites not normally accessible, but when Akt recruited to mem/ binds PIP3 via PH, conf shift-> Pi site available to PDK1, activated. Hence Akt= coincidence detector of PDK1 activity+ PIP3 production (Akt also important in ctrling cell growth+ prolif).
Akt activation in insulin signalling
Akt activation increases translocation GluT4-> PM, increases glucose uptake. Akt activation independently of upstream signalling sufficient to cause GluT4 vesicle translocation+ fusion to mem. Mech of action unclear. One mech may involve AS160- a RabGAP that increases GTP hydrolysis in specific Rab proteins, inactivating them. Pi of AS160 by Akt inhibits it, increasing Rab activity, so may provide link between Akt+ vesicle traffic. Other proteins involved and full mech TBC.
PI3K independent mech also present by which Insulin receptor reg GluT4 translocation. Insulin also reg other activities, has diff actions on diff cell types. Common signalling mechs found in insulin signalling include: activating Tyr kinases; scaffolds for signal transduction complexes; PI3K/PIP3; activation/ translocation and binding PIP3; Sr/Thr kinases and Pi; small GTPases.
Insulin-like signalling in C elegans
Insulin-like signalling in C elegans: feeds on microorganisms growing on rotting vegetation. Like many nematodes, has alt strategy when food becomes scarce- dauer stage (highly resistant to env changes+ starvation). Screens for mutants in dauer formation-> ID animals unable to form dauers/ form them constitutively (e.f.daf-2). Cloning+ analysis show dauer formation under insulin-like signalling and TGFbeta pathway- these signals converge on TF daf-16. Insulin-like ligands act through and insulin receptor (daf-2) and PI3K- this pathway affects many aspect of C elegans biology.
Ageing and insulin-like signalling
Ageing+ insulin-like signalling: Usual c elegans lifespan 19 days. Screens ID mutans w/ increased lifespan- e.g. age-1 which increases lifespan ~100%. When cloned, found to encode catalytic subunit/ PI3K. other insulin signalling genes also affect ageing, e.g. daf-2 mutants. Insulin-like signalling decreased-> more daf-16 activity-> longer lifespan. Suggested that this mech related to changes occurring on calorific restriction in mammals. Calorific restriction increases lifespan, as shown in mice; IRS mutants in flies and mice also live longer. Larva fate in beehives is determined to fate: limited diet-> worker, rich diet-> queen; IRS RNAi knockdown results in workers, even if on queen diet.
Lifespan is under genetic+ metabolic ctrl; insulin-like signalling= ancient regulator of animla metabolism (one of core pathways ctrling growth+ proliferation of animal cells).
