INFLATION DYMAMICS IN BRAZIL AN EMPIRICAL APPROACH

Förster resonance energy transferFrom Wikipedia, the free encyclopediaJump to: navigation, searchJablonski diagram of FRET with typical timescales indicatedFörster resonance energy transfer (FRET), fluorescence resonance energy transfer (FRET), resonance energy transfer (RET) orelectronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores).[1] A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling.[2] The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.[3]Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other.[4] Such measurements are used as a research tool in fields including biology and chemistry.FRET is analogous to near-field communication, in that the radius of interaction is much smaller than the wavelength of light emitted. In the near-field region, the excited chromophore emits a virtual photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable, since their existence violates the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism. Quantum electrodynamical calculations have been used to determine that radiationless (FRET) and radiative energy transfer are the short- and long-range asymptotes of a single unified mechanism.[5][6]Contents[hide]∙ 1 Terminology∙ 2 Theoretical basis∙ 3 Experimental co nfirmation of the Förster resonance energy transfer theory∙ 4 Methods to measure FRET efficiencyo 4.1 Sensitized emissiono 4.2 Photobleaching FRETo 4.3 Lifetime measurements∙ 5 Fluorophores used for FRETo 5.1 CFP-YFP pairso 5.2 BRETo 5.3 Homo-FRET∙ 6 Applicationso 6.1 Biology∙7 Other methods∙8 See also∙9 References∙10 External linksTerminology[edit]Förster resonance energy transfer is named after the German scientist Theodor Förster.[7] When both chromophores are fluorescent, the term "fluorescence resonance energy transfer" is often used instead, although the energy is not actually transferred by fluorescence.[8][9]In order to avoid an erroneous interpretation of the phenomenon that is always a nonradiative transfer of energy (even when occurring between two fluorescent chromophores), the name "Förster resonance energy transfer" is preferred to "fluorescence resonance energy transfer;" however, the latter enjoys common usage in scientific literature.[10] It should also be noted that FRET is not restricted to fluorescence. It can occur in connection with phosphorescence as well.[8]Theoretical basis[edit]The FRET efficiency () is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event:[11]where is the rate of energy transfer, the radiative decayrate, and the 's are the rate constants of any other de-excitation pathways.[12]The FRET efficiency depends on many physical parameters that can be grouped as follows:∙The distance between the donor and the acceptor (typically in the range of 1-10 nm)∙The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.∙The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.depends on the donor-to-acceptor separation distance with an inverse 6th power law due to the dipole-dipole coupling mechanism:with being the Förster distance of this pair of donor andacceptor, i.e. the distance at which the energy transfer efficiency is 50%.[12]The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation.[13][14]where is the fluorescence quantum yield of the donor in the absence of the acceptor, κ2 is the dipole orientation factor, is the refractive index of the medium, is Avogadro's number, and is the spectral overlap integral calculated aswhere is the normalized donor emission spectrum, and is the acceptor molar extinction coefficient.[15] The orientation factor κ is given by,Where denotes the normalized transition dipole moment of therespective fluorophore and denotes the normalized inter-fluorophore displacement. κ2 =2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to beisotropically oriented during the excited state lifetime. If either dye is fixed or not free to rotate, then κ2 =2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that κ2 = 2/3 doesnot result in a large error in the estimated energy transfer distance due to the sixth power dependence of R0 on κ2. Even when κ2 is quite different from 2/3 the error can be associated with a shift in R0 and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case 0 ≤ κ2≤ 4.[15]The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:[16]where and are the donor fluorescence lifetimes in the presenceand absence of an acceptor, respectively, or aswhere and are the donor fluorescence intensities with and without an acceptor, respectively.Experimental confirmation of the Förster resonance energy transfer theory[edit]The inverse sixth-power distance dependence of Förster resonance energy transfer was experimentally confirmed by Wilchek, Edelhoch and Brand[17][18] using tryptophyl peptides. Stryer, Haugland and Yguerabide[19] also experimentally demonstrated the theoretical dependence ofFörster resonance ene rgy transfer on the overlap integral by using a fused indolosteroid as a donor and a ketone as an acceptor. However, a lot of contradictions of special experiments with the theory was oserved. The reason is that the theory has approximate character and gives overstimated distances of 50-100 Angstrems (Vekshin N.L. Energy Transfer in Macromolecules, SPIE, 1997; Vekshin N.L. Photonics of Biopolymers, Springer, 2002).Methods to measure FRET efficiency[edit]In fluorescence microscopy, fluorescence confocal laser scanning microscopy, as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein–DNA interactions, and protein conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor. The FRET efficiency is measured and used to identify interactions between the labeled complexes. There are several ways of measuring the FRET efficiency by monitoring changes in the fluorescence emitted by the donor or the acceptor.[20]Sensitized emission[edit]One method of measuring FRET efficiency is to measure the variationin acceptor emission intensity.[14] When the donor and acceptor are in proximity (1–10 nm) due to the interaction of the two molecules, the acceptor emission will increase because of the intermolecularFRET from the donor to the acceptor. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed. If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection.Photobleaching FRET[edit]FRET efficiencies can also be inferred from the photobleaching rates of the donor in the presence and absence of an acceptor.[14] This method can be performed on most fluorescence microscopes; one simply shines the excitation light (of a frequency that will excite the donor but not the acceptor significantly) on specimens with and without the acceptor fluorophore and monitors the donor fluorescence (typically separated from acceptor fluorescence using a bandpass filter) over time. The timescale is that of photobleaching, which is seconds to minutes, with fluorescence in each curve being given bywhere is the photobleaching decay time constant and depends on whether the acceptor is present or not. Since photobleaching consists in the permanent inactivation of excited fluorophores, resonance energy transfer from an excited donor to an acceptor fluorophore prevents the photobleaching of that donor fluorophore, and thus high FRET efficiency leads to a longer photobleaching decay time constant:where and are the photobleaching decay time constants of thedonor in the presence and in the absence of the acceptor, respectively. (Notice that the fraction is the reciprocal of that used for lifetime measurements).This technique was introduced by Jovin in 1989.[21] Its use of anentire curve of points to extract the time constants can give it accuracy advantages over the other methods. Also, the fact that time measurements are over seconds rather than nanoseconds makes it easierthan fluorescence lifetime measurements, and because photobleaching decay rates do not generally depend on donor concentration (unless acceptor saturation is an issue), the careful control of concentrations needed for intensity measurements is not needed. It is, however, important to keep the illumination the same for the with- and without-acceptor measurements, as photobleaching increases markedly with more intense incident light.Lifetime measurements[edit]FRET efficiency can also be determined from the change in the fluorescence lifetime of the donor.[14] The lifetime of the donor will decrease in the presence of the acceptor. Lifetime measurements of FRET are used in Fluorescence-lifetime imaging microscopy.Fluorophores used for FRET[edit]If the linker is intact, excitation at the absorbance wavelength of CFP (414nm) causes emission by YFP (525nm) due to FRET. If the linker is cleaved by a protease, FRET is abolished and emission is at the CFP wavelength (475nm).CFP-YFP pairs[edit]One common pair fluorophores for biological use is a cyan fluorescent protein (CFP) – yellow fluorescent protein (YFP) pair.[22] Both are color variants of green fluorescent protein (GFP). Labeling with organic fluorescent dyes requires purification, chemical modification, and intracellular injection of a host protein. GFP variants can be attached to a host protein by genetic engineering which can be more convenient. Additionally, a fusion of CFP and YFP linked by a protease cleavage sequence can be used as a cleavage assay.[23]BRET[edit]A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor or to photobleaching. To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed.[24] This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than CFP to produce an initial photon emission compatible with YFP.Homo-FRET[edit]In general, "FRET" refers to situations where the donor and acceptor proteins (or "fluorophores") are of two different types. In many biological situations, however, researchers might need to examine the interactions between two, or more, proteins of the same type—or indeed the same protein with itself, for example if the protein folds or forms part of a polymer chain of proteins[25] or for other questions of quantification in biological cells.[26]Obviously, spectral differences will not be the tool used to detect and measure FRET, as both the acceptor and donor protein emit light with the same wavelengths. Yet researchers can detect differences in the polarisation between the light which excites the fluorophores andthe light which is emitted, in a technique called FRET anisotropy imaging; the level of quantified anisotropy (difference in polarisation between the excitation and emission beams) then becomes an indicative guide to how many FRET events have happened.[27]Applications[edit]Biology[edit]FRET has been used to measure distance and detect molecular interactions in a number of systems and has applications in biology and chemistry.[28] FRET can be used to measure distances between domains in a single protein and therefore to provide information about protein conformation.[29] FRET can also detect interaction between proteins.[30] Applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including intergrins and membrane proteins.[31] FRET can be used to obtain information about metabolic or signaling pathways.[32] FRET is also used to study lipid rafts in cell membranes.[33]FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors.The applications of Fluorescence Resonance Energy Transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. FRET can be used as spectroscopic ruler in various areas such as structural elucidation of biological molecules and their interactions in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterial etc. Based on the mechanism of FRET a variety of novel chemical sensors and biosensors have been developed.[34]Other methods[edit]A different, but related, mechanism is Dexter Electron Transfer.An alternative method to detecting protein–protein proximity is the bimolecular fluorescence complementation (BiFC) where two halves of a YFP are fused to a protein. When these two halves meet they form a fluorophore after about 60 s – 1 hr.[35]See also[edit]∙Förster coupling∙Surface energy transfer∙Dexter electron transfer∙Time-resolved fluorescence energy transferReferences[edit]1.Jump up ^ Cheng, Ping-Chin (2006). "The Contrast Formation in OpticalMicroscopy". In Pawley, James B. 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ReviewThe tumor suppressor kinase LKB 1:lessons from mouse modelsSaara Ollila and Tomi P.Ma¨kela ¨*Institute of Biotechnology,University of Helsinki,Viikki Biocenter,Viikinkaari 9B,FIN-00014,Helsinki,Finland*Correspondence to:Tomi P.Ma¨kela ¨,E-mail:tomi.makela@helsinki.fiMutations in the tumor suppressor gene LKB 1are important in hereditary Peutz–Jeghers syndrome,as well as in sporadic cancersincluding lung and cervical cancer.LKB 1is a kinase-activating kinase,and a number of LKB 1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism,polarity,cytoskeleton organization,and prolifer-ation.Conditional targeting approaches are beginning to demonstrate the relevance and specificity of these signaling pathways in development and homeostasis of multiple organs.More than one of the pathways also appear to contribute to tumor growth fol-lowing Lkb 1deficiencies based on a number of mouse tumor models.Lkb 1-dependent activation of AMPK and subsequent inacti-vation of mammalian target of rapamycin signaling are implicated in several of the models,and other less well characterized pathways are also involved.Conditional targeting studies of Lkb 1also point an important role of LKB 1in epithelial–mesenchymal interactions,significantly expanding knowledge on the relevance of LKB 1in human disease.Keywords:LKB 1,tumor suppressor,mouse model,AMPKIntroductionCancer arises as a result of accumulating genetic and epige-netic changes,which compromise the cell’s ability to control its identity and proliferation.Many identified tumor suppressors play a well-established role in regulation of cell growth and div-ision (e.g.Rb,APC,p 21,PTEN)and genome maintenance (e.g.p 53,BRCA 1-2,ATM,ATR,MLH 1,MSH 2),providing a logical link between the loss of gene product and promotion of carcinogen-esis.An interesting exception is the serine /threonine kinase gene LKB 1(also known as STK 11),which has in recent years taken a prominent position among tumor suppressors.Heterozygous germline mutations in LKB 1predispose to Peutz–Jeghers syndrome (PJS)where patients develop benign polyps in the gastrointestinal (GI)tract and are in high risk of developing malignant tumors in GI tract,breast,and gyneco-logical organs (Giardiello et al .,2000).Importantly,somatic LKB 1mutations are found at least in lung (Ji et al.,2007)and cervical cancer (Wingo et al .,2009).Through phosphoryl-ation of several cellular kinases LKB 1has been implicated in control of cellular and organismal metabolism,cell polarity,and a variety of other functions ranging from proliferation and migration to senescence,apoptosis,DNA damage responseand differentiation (Vaahtomeri and Ma¨kela ¨,2011).Despite these many functions attributed to LKB 1,their specific contri-butions to the maintenance of tissue homeostasis in vivo and tumor growth are only sketchily appearing with thedevelopment of LKB 1mouse models.This work is important to enable rational treatment strategies to LKB 1-deficient tumors.The LKB 1kinase acts in a trimer with a pseudokinase STRAD and the scaffold protein MO 25to phosphorylate at least 14kinases with conserved activation sites (Katajisto et al.,2007).A well-known substrate of LKB 1is AMPK,which is the master reg-ulator of cellular and organismal metabolism,providing a putative downstream pathway to LKB 1-mediated tumor suppression (Shackelford and Shaw,2009).In mouse studies,AMPK requires LKB 1for activation in vivo in most tissues (Sakamoto et al .,2005;Shaw et al .,2005;Contreras et al .,2008;Hezel et al .,2008).AMPK senses the energy state of cells through monitoring AMP levels as a sensitive readout for ATP.AMPK is activated following exercise,hypoxia,or glucose deprivation,after which it phosphor-ylates multiple targets to increase energy uptake and catabolic processes such as glucose uptake and fatty acid oxidation,and suppress anabolic processes such as lipogenesis and cholesterol synthesis (Hardie et al.,2003).AMPK is the potential candidate to mediate LKB 1’s effects in cell growth via the mammalian target of rapamycin (mTOR)signal-ing (Corradetti et al .,2004;Shaw et al .,2004),which is the pathway monitoring the availability of nutrients in regulation of cell size and protein synthesis as well as proliferation (Zoncu et al.,2011).Increased mTOR signaling is common in cancer (Guertin and Sabatini,2007)and also present in at least some Lkb 1-deficient tumors (Shaw et al.,2004;Ji et al.,2007;Contreras et al.,2008;Hezel et al.,2008;Shackelford et al.,2009).An additional link between LKB 1and mTOR pathway#The Author (2011).Published by Oxford University Press on behalf of Journal ofMolecular Cell Biology ,IBCB,SIBS,CAS.All rights reserved.doi:10.1093/jmcb /mjr 016Journal of Molecular Cell Biology (2011),Vol no.0,1–11|1Journal of Molecular Cell Biology Advance Access published September 15, 2011 at Shihezi University on September 27, 2011 Downloaded frommay lie in regulation of PI 3K-Akt pathway inhibitor PTEN by LKB 1(Mehenni et al .,2005).Loss of cell polarity is commonly noted in cancer,and LKB 1is an important factor for cell polarity in different organisms.In C.elegans ,the orthologs for LKB 1(par-4)and MARK s (par-1)were identified in a panel of six partitioning (par )mutants which disrupted the polarity of the early embryos (Kemphues et al.,1988).In Drosophila ,Lkb 1is required for proper oocyte polarity (Martin and St Johnston,2003).In mammalian cells,in both 2D and 3D cell culture models and in vivo ,LKB 1is known to regulate polarity (Baas et al .,2004;Partanen et al .,2007;Hezel et al .,2008).Polarity defects are,however,not seen in all Lkb 1-deficient tumors (Contreras et al.,2008,2010).Several of the LKB 1substrates have been reported to mediate the regulation of cell polarity through regulating the cytoskeleton and formation of cell–cell junctions.MARK kinases are implicated in the stability of microtubules by phosphorylating and thereby dissociation microtubule-associated proteins (MAPs),for example the tau protein,from microtubules (Drewes et al .,1997;Stoothoff and Johnson,2005).Neuronal polarity and axon formation are regu-lated by LKB 1at least partially via BRSK kinases (Kishi et al.,2005;Barnes et al.,2007).To what extent LKB 1acts as a polarity protein in mammalian non-neuronal cells still remains to be deter-mined,although at least in both exo-and endocrine pancreas Lkb 1loss leads to polarity defects in vivo (Hezel et al .,2008;Granot et al .,2009).As formation of stress fibers is essential incell contractility,recent studies associate LKB 1with cell motility via NUAK 1and NUAK 2,which have been implicated in regulation of myosin light chain phosphorylation (Vallenius et al.,2010;Zagorska et al.,2010).For detailed information of the molecular signaling pathways of Lkb 1,the reader is recommended recent reviews more focused on that topic (Katajisto et al.,2007;Hezel and Bardeesy,2008;Vaahtomeri and Ma¨kela ¨,2011).Role of Lkb 1in development and tissue homeostasis in miceAlthough LKB 1is a tumor suppressor,inactivation of Lkb 1through homologous recombination or ‘knock-out’(KO)does not always lead to tumors.This is due partly to essential functions of Lkb 1in development and partly demonstrates the tissue-specificity of Lkb 1functions,where in some cell types biallelic deletion is detrimental to cells or affects specific functions in metabolism as summarized in Figure 1and discussed below.Role of Lkb 1in embryogenesisGeneration of full KO revealed that Lkb 1is essential for embry-ogenesis;no viable Lkb 12/2embryos were seen after E 11.Analysis of the E 8.5–E 9.5embryos revealed severe developmen-tal defects including impaired neural tube closure and somitogen-esis,mesenchymal tissue cell death,and defective vasculature.The extra-embryonic tissues (yolk sac and placenta)were also deformed.VEGF signaling was highly upregulated in theKOFigure 1Non-tumorigenic phenotypes following Lkb 1targeting in mice.Phenotypes (green)are grouped according to tissue type,cell typeaffected /analyzed (blue),and alleles used for targeting.When appropriate,activator of deletor is indicated in purple.Noted signaling change(s)indicated in red.Alleles as displayed in original publications except for Lkb 1flox 2h /flox 2h hypomorphic Lkb 1(Sakamoto et al,2005).(1)Londesborough et al.,2008;(2)Ohashi et al.,2010;(3)Cao et al.,2010;Tamas et al.,2010;(4)Shorning et al.,2009;(5)Woods et al.,2011;(6)Shaw et al.,2005;(7)Sun et al.,2010a ;(8)Sun et al.,2011;(9)Granot et al.,2009;Fu et al.,2009;(10)Koh et al.,2006;(11)Sakamoto et al.,2005;(12)Sakamoto et al.,2006;Jessen,et al.,2010;(13)Ikeda et al.2009;(14)Gurumurthy et al.,2010;Nakada et al.,2010;(15)Gan et al.,2010;(16)Barnes et al.,2007;(17)Ylikorkala et al.,2001.tam,tamoxifen;b -NF,b -naphtoflavone;pIpC,polyinosinic–polycytidylic acid;iv,intravenous.2|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromembryos,possibly relating to the vascular phenotype (Ylikorkala et al .,2001).Embryonic lethality,no embryonic turning,and small somites were also shown in another report of Lkb 1full KO (Jishage et al .,2002).The severe developmental defect was not a result of the abnormal extra-embryonic tissues,since epiblast-specific conditional inactivation of Lkb 1using Mox 1-Cre resulted in very similar embryonic lethal phenotype to full KO (Londesborough et al .,2008).The important role of Lkb 1in development and maintenance of neurons,mesenchymal cells,and vascularization has been recapitulated in tissue-specific Lkb 1KOs.Role of Lkb 1in angiogenesisLondesborough et al .(2008)further dissected the role of Lkb 1in endothelia by deleting Lkb 1in vascular endothelial cells using Tie 1-Cre (Figure 1).The mice died at E 12.5and displayed dilated embryonic vessels and pericardial swelling.The vessels were irre-gular and distorted and suffered from inadequate supportive vas-cular smooth muscle cell layer.Since Tgf b signaling was reduced both in Lkb 1-deficient mouse yolk sacs and human umbilical vein endothelial cells (HUVECs)where LKB 1expression was silenced by siRNA,the vascular phenotype was suggested to result from a loss of supporting vascular smooth muscle cells as a conse-quence of attenuated Tgf b signaling from endothelial cells (Londesborough et al .,2008).Another report also described mice lacking Lkb 1in endothelial cells,deleted using Tie 2-Cre driver (Ohashi et al.,2010)(Figure 1).This study repeated the finding that endothelial Lkb 1is essential for proper embryonic development and no homozygous mutants were born.Analysis of heterozygous Tie 2-Cre;Lkb 1flox /+mice revealed that the mice,including vasculature,seemed phenotypically normal,but displayed reduced revascularization after hind-limb ischemia.Studies in mouse tissues,primary mouse endothelial cells,and HUVECs implemented that the phenotype was mediated via AMPK (Ohashi et al.,2010).In this study,the authors did not address the contribution of Tgf b signaling to the observed phenotype.In the Tie 2-Cre model,Lkb 1–AMPK axis seemed to mediate proangiogenetic signaling as Lkb 1heterozygosity resulted in reduction of revascularization in adult mice (Ohashi et al.,2010).In developing embryo,increased VEGF signaling upon Lkb 1loss would suggest the opposite,antiangiogenic role for Lkb 1(Ylikorkala et al .,2001).Also in the context of PJS polyps where a loss of Lkb 1leads to increased HIF 1a and vasculature,Lkb 1seems to be rather antiangiogenic (Shackelford et al .,2009).However,reduced capillary density was reported in mice where Lkb 1was conditionally deleted from the heart (Ikeda et al .,2009).In 3D culture system where endothelial cells are embedded in Matrigel,both over-expression (Xie et al .,2009)and inhibition (Ohashi et al.,2010)of Lkb 1have been reported to inhibit network formation,suggesting proper expression of LKB 1is essential for angiogenesis.Thus,the precise role of Lkb 1in angiogenesis seems to be dependent on the tissue type and /or the developmental phase,varying from inhibition to promotion.Role of Lkb 1in liverThe finding that Lkb 1functions upstream of AMPK (Shaw et al .,2004)led to interest to study its effects in liver,where many path-ways of carbohydrate and lipid metabolism,including glycogen-esis,glycogenolysis,gluconeogenesis,lipogenesis,and cholesterol synthesis take place.Tail-vain injection of Adeno-Cre to mice carrying conditional Lkb 1allele led to hepatocyte-specific Lkb 1deletion since Adeno-Cre has high tropism for hepatocytes (Shaw et al .,2005)(Figure 1).Lkb 1loss resulted in nearly complete abolishment of AMPK activation in liver,and the glucose metabolism of the mice was impaired demonstrated by elevated blood glucose.CRTC 2phosphorylation was reduced in the livers of the mice,leading to elevated CREB-mediated transcription,including expression of PGC 1a and other gluconeogenetic genes.Also lipogenetic genes were over-expressed.Metformin,the diabetes drug which reduces blood glucose levels via AMPK pathway (Zhou et al .,2001),did not lower blood glucose in the liver-specific Lkb 1KO mice,demonstrating that AMPK activity induced by Lkb 1in liver is required for the effects of metformin in vivo .In another report of liver-specific Lkb 1knockout using Alb-Cre driver,Woods et al.(2011)reported defective bile ducts in liver,leading to accumulation of bile in liver and serum (Figure 1).Bile salt export pump was not located in canalicular membrane of the bile canaliculi,indicating possible defects in cell polarity.The mice also suffered from cholestasis (Woods et al.,2011).These reports of liver-targeted deletions of Lkb 1demonstrate the critical requirement of Lkb 1in glucose,lipid,bile,and cholesterol metab-olism.Furthermore,they show that in liver,Lkb 1is the main acti-vator of AMPK,and its activity is required for the AMPK-mediated suppression of lipogenesis and gluconeogenesis to take place.Role of Lkb 1in muscleMuscles are highly energy-consuming tissues whose glucose homeostasis needs to be regulated both in response to insulin after blood sugar increase,and to exercise-mediated deficiency of glucose storage.Sakamoto et al.(2005)provided the first genetic evidence that Lkb 1is required for AMPK activation in vivo in skeletal muscle.They generated conditional Lkb 1mice in which cDNA of Lkb 1exons 5–7fused with neomycin resistance cassette,surrounded by loxP sites,was inserted between exons 4and 8in the genomic Lkb 1locus.The resulting mice were hypomorphic and expressed only 10%–20%of normal levels of Lkb 1in the absence of Cre -mediated ing MCK-Cre driver to create muscle-specific Lkb 1KO,they found that AMPK a 2(one of the two alternative catalytic subunits of AMPK)activation either by the AMP analog AICAR,muscle con-traction or phenformin,a similar blood glucose lowering drug to metformin,was lost and AMPK a 1activation greatly reduced.Upon contraction,glucose transport to muscle cells was abol-ished (Sakamoto et al.,2005).In another study using the same muscle-specific MCK-Cre with another (non-hypomorphic)con-ditional Lkb 1line,effects of Lkb 1loss in muscle to levels of blood glucose were investigated (Koh et al.,2006)(Figure 1).Interestingly,glucose metabolism seemed to be enhanced in these mice,demonstrated by reduced fasting blood glucose and blood insulin concentrations,improved glucose tolerance,and increased muscle glucose uptake.This phenotype,indicating that Lkb 1in muscle functions as a negative regulator of glucose metabolism,was suggested to be resulting from improved muscle glucose uptake,mediated by increased phosphorylation of Akt and reduced the gene expression of the Akt inhibitor TRB 3.Lkb 1loss abolished the activity of AMPK a 2,but notLessons from LKB 1mouse modelsJournal of Molecular Cell Biology |3at Shihezi University on September 27, 2011 Downloaded fromAMPK a 1in muscle cells.Also MARK 4,but not MARK 2/3activitywas significantly reduced.Based on this study,the metabolic effects mediated by Lkb 1in muscle seem to oppose those of the liver,at least in terms of blood glucose levels (Koh et al.,2006).Recently,the Lkb 1substrate NUAK 2was proposed to be a mediator of contraction-stimulated glucose transport by skel-etal muscle (Koh et al.,2010).Also cardiac muscle lacking Lkb 1has been investigated.Sakamoto et al.(2006)studied the effect of Lkb 1deficiency in heart using the MCK-Cre driver,which deletes Lkb 1in both skel-etal and cardiac myocytes and found that Lkb 1inactivation did not lead to overt cardiac dysfunction,although the weight of the heart was reduced and the atria enlarged;however,the study revealed that cardiac Lkb 1is required for activation of AMPK a 2both in basal conditions and in response to ischemia (Figure 1).Also Jessen et al.(2010)used the MCK-Cre driver but the Lkb 1allele was not hypomorphic as in the Sakamoto et al.(2006)study.They showed that ablation of Lkb 1in heart leads to impaired cardiac function both in basic conditions and post-ischemia and suggested that failure to downregulate mTOR sig-naling by AMPK a 2activation underlined the phenotypes.Ikeda et al.(2009)used a -MHC-Cre to delete Lkb 1specifically from the heart,and a more severe phenotype was observed:the mice displayed hypertrophy and impaired function of the heart,reduction of cardiac capillary density,and increased fibrosis and collagen content and died by 6months of age.The differ-ences between these phenotypes may reflect differences in the timing of Cre activity,specificity of the Cre recombination,and /or the conditional Lkb 1allele used.However,it seems clear that Lkb 1is needed for the normal function of heart both in basal and ischemic conditions.Role of Lkb 1in pancreasPancreatic b -cells secrete insulin and are thus important mediators of whole-body glucose metabolism.As Lkb 1–AMPK axis is important in regulation of liver metabolism and muscle glucose homeostasis,it is of interest to study whether Lkb 1has an effect on the insulin release.Granot et al.(2009)used the Pdx 1-CreER driver to delete pancreatic Lkb 1in 6-week-old mice by tamoxifen injection (Figure 1).In response to glucose injection,the mutant mice secreted more insulin than control mice,which carried the conditional Lkb 1allele but were not subjected to tamoxifen injection.Deletion of Lkb 1led to increased size of b -cells together with disrupted polarity.Increased mTOR signal-ing seemed to mediate the cell size increase,while the polarity defect took place at least partially through MARK 2.Increased insulin secretion was partially dependent on AMPK (Granot et al .,2009).Fu et al .(2009)used the same Pdx 1-CreER system to delete Lkb 1in adult b -cells and also found that the mice showed improved glucose tolerance,b -cells mass had increased,and mTOR pathway was activated (Figure 1).These results place Lkb 1as an important regulator of pancreatic b -cell size,polarity,and function,further highlighting its essence in regulation of organismal metabolism.Sun et al.(2010a)investigated pancreatic b -cells with the Rip 2-Cre driver,which activates Cre -mediated recombination in pancreatic b -cells and some hypothalamic neurons,and found that the mice displayed diminished food intake and weight gain,enhanced insulin secretion,and improved glucose tolerance (Figure 1).Also here,mTOR pathway was activated.However,the study by the same group where both AMPK a subunits were deleted in b -cells using the same Rip 2-Cre showed decreased insulin secretion (Sun et al.,2010b ).This suggests that Lkb 1loss regulates mTOR signaling in b -cells partially independent of AMPK,or that the hypothalamic Lkb 1and AMPK have different functions,impacting the feeding behavior and hormonal balance.Role of Lkb 1in immune systemThree recent studies elegantly demonstrated that Lkb 1regu-lates the quiescence and maintenance of hematopoietic stem cells (HSCs)using conditional Lkb 1alleles with Mx 1-Cre followed by injections of polyinosinic–polycytidylic acid (pIpC),or Rosa 26-CreERt 2followed by tamoxifen injections (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010)(Figure 1).Both approaches resulted in a similar phenotype:increased pro-liferation followed soon by decline in HSC number,resulting in loss of all immune cell types (pancytopenia)and death.Transplantation experiments demonstrated that Lkb 1-deficient HSCs were not able to reconstitute the bone marrow of irradiated wild-type (wt)mice,nor were they able to compete with wt donor cells,demonstrating that the effect was cell-autonomous;mito-chondrial defects and decreased ATP levels,as well as altered long-chain fatty acid and nucleotide metabolite levels suggested metabolic defects to underlie the phenotypes noted (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).Interestingly,only minor similarities in mitochondrial phenotypes were found when mice defective for both AMPK a subunits were compared with Lkb 1KO mice (Nakada et al.,2010),implicating other Lkb 1substrates in these phenotypes.Consistent with this,rapamycin or AMPK activators AICAR and A 769662did not rescue the phenotype in any of the studies.Immune cell apopto-sis was increased,and Lkb 1-deficient HSCs also demonstrated increased autophagy in bone marrow,and inhibiting this further decreased immune cell survival (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).This would suggest that Lkb 1in this context is suppressing autophagy,whereas previously it has been reported to activate it following elevation of reactive oxygen species (Alexander et al.,2010).Yet another phenotype potentially decreasing HSC viability was the noted increase in supernumerary centrosomes,aberrant mitotic spindles,and aneuploidy (Nakada et al.,2010),which could be due to compro-mised BRSK 2activity (Alvarado-Kristensson et al.,2009).Recently,two groups generated mice where Lkb 1expression is specifically abolished in the T cell progenitors using the proximal p 56lck-Cre promoter.The studies demonstrate severe deficiency in survival and proliferation of T cell progenitors and mature T cells in the absence of Lkb 1(Cao et al.,2010;Tamas et al.,2010)(Figure 1).Also the survival of isolated peripheral T cells in vitro was dependent on Lkb 1(Tamas et al.,2010).Transfection of thymo-cytes with constitutively active AMPK a 2partially rescued the thy-mocytes from cell death,indicating that thymocyte survival is mediated at least via AMPK pathway (Cao et al.,2010).Thus,the common hematopoietic cell precursors and T cell precursors seem to have different requirement for AMPK signaling,although cell sur-vival is defective in both cell types in the absence of Lkb 1.The studies in hematopoietic cells have revealed an interesting aspect4|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromof Lkb 1biology:although being a tumor suppressor in some tissues,in others Lkb 1is required for survival.Role of Lkb 1in nervous systemLkb 1KO embryos exhibit severe deficiencies in development of neuronal tissues (Ylikorkala et al .,2001).Since LKB 1orthologs in nematodes and fruit flies have been identified through their indis-pensable role in establishing polarity (Kemphues et al .,1988;Martin and St Johnston,2003)and LKB 1regulates polarity also in some mammalian cells (Baas et al .,2004;Partanen et al .,2007),it was of interest to generate models which would reveal the in vivo relevance of Lkb 1in establishing the axon-dendrite polarity in neuronal cells.Barnes et al.(2007)deleted Lkb 1in cer-ebral cortex of developing mice using Emx-Cre driver and showed that Lkb 1and its substrates BRSK 1and BRSK 2are required for axon specification in the studied neurons.This finding confirmed the previously described role of BRSK kinases in neuronal polar-ization (Kishi et al .,2005),and placed Lkb 1as the upstream kinase required for the polarization to take place.Lkb 1-activated BRSKs were shown to modify the cytoskeleton by phosphorylating MAPs (Barnes et al.,2007).Studies in rat hip-pocampal neurons in vitro and developing rat cortical neurons in vivo agreed with the finding that Lkb 1is essential in establishing neuronal polarity;there,lack of either Lkb 1or STRAD prevented axon differentiation (Shelly et al.,2007).Interestingly,over-expression of Lkb 1and STRAD resulted in formation of multiple axons.PKA-mediated phosphorylation of Lkb 1Ser 431was shown to be required for the axon specification (Barnes et al.,2007;Shelly et al.,2007).Thus,Lkb 1activity is modulated by upstream factors in a tissue-and context-specific manner.Not only axon specification but also maintenance seems to be regulated via Lkb 1in some systems.Sun et al.(2011)reported,using the pancreatic and hypothalamic Rip 2-Cre ,that the mice developed hind-limb paralysis due to axon degeneration in thor-acic spinal cord neurons at about 7–8weeks of age (Figure 1).The Rip 2-Cre was found to be active also in spinal cord,especially in the thoracic segments.Deleting both AMPK a subunits did not result in axon degeneration or paralysis,and the authors specu-lated that in the absence of Lkb 1,the neuronal polarization and axon degeneration defects might be mediated by BRSK kinase pathways (Sun et al.,2011).PJS and its mouse modelsLKB 1was linked to human disease when its mutations were found to be causative for PJS (Hemminki et al .,1998;Jenne et al .,1998).A major manifestation in PJS is the appearance of large occluding hamartomatous polyps in the GI tract (Giardiello and Trimbath,2006).Mice carrying one inactivated allele of Lkb 1(Lkb 1+/2)recapitulate PJS by developing hamartomatous GI polyps which are indistinguishable from PJS patient polyps (Bardeesy et al .,2002;Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)(Figure 2),although in mice polyps appear more in the stomach and less in the small intestine.Polyps appear at 4–6months (Udd et al.,2010),and lead to lethality at an average age of 11months due primarily to obstructions.Biallelic loss of wt Lkb 1is not a prerequisite for polyp formation,indicating that Lkb 1is a haploinsufficient tumor suppressor at least in the context of PJS polyps (Jishage et al .,2002;Miyoshiet al .,2002;Rossi et al .,2002).Strong up-regulation of COX 2has been identified in the mouse and also PJS patient polyps (Rossi et al .,2002),and COX 2inhibitors have been shown to be efficient suppressors of PJS polyps (Udd et al .,2004).PJS is associated with elevated risk of cancer,especially in the GI tract,and also in breast,pancreas and gynecological cancers (Giardiello and Trimbath,2006;Hearle et al .,2006;Mehenni et al .,2006).Lkb 1+/2mice in turn have been reported to have increased frequency of cancer in liver (Nakau et al .,2002),bones (Robinson et al .,2008),and endometrium (Contreras et al .,2008)(Figure 2).Polyposis in Lkb 1+/2mice is accelerated in a p 53-deficient background (our unpublished data)(Wei et al .,2005;Takeda et al .,2006)(Figure 2),and p 53mutations are detected in the GI cancers of PJS patients (Miyaki et al .,2000).Despite these observations,progression of the benign hamarto-matous polyps to dysplasia or carcinoma is not clearly estab-lished possibly due to the rapid growth of the hamartomatous polyps leading to GI occlusions.As haploinsufficiency of Lkb 1is sufficient for polyp initiation (Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)though biallelic loss has been noted (Bardeesy et al .,2002),loss of the remaining allele of Lkb 1may represent a progression step,although it has also been suggested that the loss of Lkb 1is associated with the resist-ance to progression in this context (Bardeesy et al.,2002).Mesenchymal Lkb 1loss leads to PJS-type polyposis in mice PJS polyps are classified as hamartomatous polyps thought to contain all the cell types of the surrounding tissue.However,it was recently noted that epithelial differentiation is disrupted in gastric and small intestinal polyps in Lkb 1+/2mice (Udd et al.,2010),but the model did not enable distinguishing whether this was a cell autonomous function of Lkb 1in epithelial cells.Biallelic disruption of Lkb 1in GI epithelia lead to imbalanced differentiation and positioning of epithelial cells (Shorning et al .,2009)(Figure 1),but was not reported to be associated with tumorigenesis.Polyps in both PJS patients and Lkb 1+/2mice harbor a large component of smooth muscle tissue.Remarkably,in a mouse model,where Lkb 1deficiency was restricted to the smooth muscle lineage by using a tamoxifen-inducible SM 22-CreERt 2line,PJS type polyps appeared in stomachs of the mice with the hetero-and homozygous Lkb 1mutants (Katajisto et al .,2008)(Figure 2).The polyps appeared later than those in the Lkb 1+/2mice,suggesting either that tamoxifen-induced Lkb 1loss at 6weeks of age delayed the poly-posis,or that mesenchymal loss of Lkb 1signaling is sufficient to drive hyperproliferation of epithelial tissue,but that coexisting epithelial mutations accelerate the process.This interesting aspect of Lkb 1signaling in tissue interactions is discussed later.Other Lkb 1tumor mouse modelsInactivating LKB 1mutations are associated with the develop-ment of cancer in several tissues.Various strategies of targeted inactivation of Lkb 1in mice,sometimes in combination of other tumorigenic mutations,have led to the development of various types and grades of tumors in multiple tissues,sometimes mod-eling human cancers in very useful ways as discussed below and summarized in Figure 2.Lessons from LKB 1mouse modelsJournal of Molecular Cell Biology |5at Shihezi University on September 27, 2011 Downloaded from。

国际免疫学杂志2021年1月第44卷第1期丨m j hnmUn 〇U a n .2021,V 〇1.44，N 〇. 1• 71 ••综述•心肌缺血再灌注损伤中细胞焦亡的研究进展刘成兴林吉斌李大主华中科技大学同济医学院附属协和医院心内科，武汉43〇〇22 通信作者：李大主，E m a i l :lid a z h u h p @ s o h u . c o m ,电话：139****1110【摘要】细胞焦亡是近年来发现并被证实的一种新的细胞程序性死亡方式，它的特征是依赖半胱氨酸天冬氨酸酶丨（cysteinyl aspartate specific proteinase 1 ,caspase-l)并伴随大量炎症因子的释放。

细胞 焦亡参与了包括感染性疾病在内的多种疾病的病理生理过程作为一种新的调节性细胞死亡方式，近 年来细胞焦亡受到了广泛的关注。

新近研究发现，细胞焦亡也参与了心肌缺血再灌注损伤（myocardialiscliemia reperfusion injury,MIRI)过程，文摩:就M 丨R I 中细胞焦亡的研究进展进行综述.【关键词】细胞焦亡;心肌缺血再灌注损伤;炎性小体基金项目：国家自然科学基金（8〗670404,81700390)D O I ：10. 3760/cma.j. issn. 16734394.2021.01.012Research progress of cell pyroptosis in myocardial ischemia reperfusion injuryLiu Chengxing，Lin Jibin ,Li DazhuDepartment of Cardiology .Union Hospital ,Tongji Medical College ,Haazhong University o f Science and Technolo­gy ,Wuhan 430022, ChinaCorresponding author ： Li I)azhu , Email ： lidazkuhp@ sohu. com , Tel ： 13971091 1 10【Abstract 】 Cell pyroptosis is a newly found m o d e of regulated cell death and experimentally verified re-c-ently. I t i s characterized l )y i t s dependence on cysteinyl asparlale specific proteinase 1 (caspase-1 ) and the re­lease of a large numb e r of inflammatory factors. Pyroptosis i s involved in the pathophysiological process of m a n y diseases including infectious diseases. As a new way of regulatory cell death, pyroptosis has attracted extensive attention in recent years. Recent studies have found that i t participates iq the pathological processes of myocardi­al ischemia reperfusion injury ( M I R I ) . This review summarized the research progress of cell pyroptosis in MIRI.【Key words 】Cell pyroptosis; Myocardial ischemia reperfusion injury; InflammasomeFund program ： National Natural Science Foundation of China( 81670404,81700390)D O I ： 10. 3760/cma. j . issn. 16734394. 2021.01.012上的模式识别受体（pattern recognition receptor ,P R R)识别并诱导相应的炎症反应过程，这些炎症细胞包括单核/巨噬细胞、中性粒细胞和树突状细胞 等夂。

2 ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２３．０１．０２８细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用刘天会首都医科大学附属北京友谊医院肝病中心，北京１０００５０通信作者：刘天会，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）摘要：细胞器除了具有各自特定的功能外，还可与其他细胞器相互作用完成重要的生理功能。

细胞器之间相互作用的异常与疾病的发生发展密切相关。

近年来，细胞器之间相互作用在非酒精性脂肪性肝病（ＮＡＦＬＤ）发生发展中的作用受到关注，特别是线粒体、脂滴与其他细胞器之间的相互作用。

关键词：非酒精性脂肪性肝病；细胞器；线粒体；脂肪滴基金项目：国家自然科学基金面上项目（８２０７０６１８）ＲｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅＬＩＵＴｉａｎｈｕｉ．（ＬｉｖｅｒＲｅｓｅａｒｃｈＣｅｎｔｅｒ，ＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌ，ＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１０００５０，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＬＩＵＴｉａｎｈｕｉ，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）Ａｂｓｔｒａｃｔ：Ｉｎａｄｄｉｔｉｏｎｔｏｉｔｓｏｗｎｓｐｅｃｉｆｉｃｆｕｎｃｔｉｏｎｓ，ａｎｏｒｇａｎｅｌｌｅｃａｎａｌｓｏｉｎｔｅｒａｃｔｗｉｔｈｏｔｈｅｒｏｒｇａｎｅｌｌｅｓｔｏｃｏｍｐｌｅｔｅｉｍｐｏｒｔａｎｔｐｈｙｓｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｓ．Ｔｈｅｄｉｓｏｒｄｅｒｓｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓａｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｖａｒｉｏｕｓｄｉｓｅａｓｅｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｔｈｅｒｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓｈａｓａｔｔｒａｃｔｅｄｍｏｒｅａｔｔｅｎｔｉｏｎｉｎｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ，ｅｓｐｅｃｉａｌｌｙｔｈｅｉｎｔｅｒａｃｔｉｏｎｓｂｅｔｗｅｅｎｍｉｔｏｃｈｏｎｄｒｉａ，ｌｉｐｉｄｄｒｏｐｌｅｔｓ，ａｎｄｏｔｈｅｒｏｒｇａｎｅｌｌｅｓ．Ｋｅｙｗｏｒｄｓ：Ｎｏｎ－ａｌｃｏｈｏｌｉｃＦａｔｔｙＬｉｖｅｒＤｉｓｅａｓｅ；Ｏｒｇａｎｅｌｌｅｓ；Ｍｉｔｏｃｈｏｎｄｒｉａ；ＬｉｐｉｄＤｒｏｐｌｅｔｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８２０７０６１８） 细胞器可以通过膜接触位点与其他细胞器相互作用，完成物质与信息的交换，形成互作网络［１］。