The initiation and propagation of an action potential through the membranous system of the sarcolemma and the tubular network lead to the activation of the Ca2+-release units (CRU): tightly coupled dihydropyridine and ryanodine (RyR) receptors. The RyR gating allows a rapid, massive, and highly regulated release of Ca2+ from the sarcoplasmic reticulum (SR). The release from triadic places generates a sarcomeric gradient of Ca2+ concentrations ([Ca2+]) depending on the distance of a subcellular region from the CRU. Upon release, the diffusing Ca2+ has multiple fates: binds to troponin C thus activating the contractile machinery, binds to classical sarcoplasmic Ca2+ buffers such as parvalbumin, adenosine triphosphate and, experimentally, fluorescent dyes, enters the mitochondria and the SR, or is recycled through the Na+/Ca2+ exchanger and store-operated Ca2+ entry (SOCE) mechanisms. To commemorate the 7th decade after being coined, we comprehensively and critically reviewed “old”, historical landmarks and well-established concepts, and blended them with recent advances to have a complete, quantitative-focused landscape of the ECC. We discuss the: 1) elucidation of the CRU structures at near-atomic resolution and its implications for functional coupling; 2) reliable quantification of peak sarcoplasmic [Ca2+] using fast, low affinity Ca2+ dyes and the relative contributions of the Ca2+-binding mechanisms to the whole concert of Ca2+ fluxes inside the fibre; 3) articulation of this novel quantitative information with the unveiled structural details of the molecular machinery involved in mitochondrial Ca2+ handing to understand how and how much Ca2+ enters the mitochondria; 4) presence of the SOCE machinery and its different modes of activation, which awaits understanding of its magnitude and relevance in situ; 5) pharmacology of the ECC, and 6) emerging topics such as the use and potential applications of super-resolution and induced pluripotent stem cells (iPSC) in ECC. Blending the old with the new works better!The excitation–contraction coupling (ECC) in skeletal muscle refers to the Ca By the 1940s, some evidence was published considering the existence of a link between the excitation and the contraction of the skeletal muscle. However, it was very scarce, fraught with technical limitations and in some cases speculative, although considered “plausible”. Alexander Sandow stated in 1952 that the muscle “dependence of contraction on excitation indicates that there must be some process that is initiated in the excited membrane and which by moving inward makes contact with the contractile elements so as to initiate contraction. We shall designate the entire sequence of reactions -excitation, inward acting link, and activation of contraction- by the term excitation-contraction (E-C) coupling” . This toSince then, we have seen the development of a huge amount of technology with increasingly improved temporal and spatial resolution, which has been applied to gain insight into the excitation-contraction coupling (ECC) in skeletal muscle and have helped us outline the current landscape of this phenomenon.Here, we present the basics of the ECC in skeletal muscle under physiological conditions, highlighting recent exciting conceptual advances and technical developments for its study. We present information gathered in vertebrate models but focused on mammalian ECC. Blending the old with the new allows us to commemorate 7 decades of hard work by honoring notable, classical contributions from many researchers in the field, but also by integrating new advances performed by intrepid young generations. We believe this is the best way to yield the most comprehensive possible picture of the ECC in skeletal muscle.th Century. Two well accepted concepts by that time were that the sarcolemma had excitatory properties (assumed to be limited only to the surface of the sarcolemma) and that soon after excited, the active state of the muscle was established , which led to the proposal of a relaxing, “Marsh-Bendall” factor in muscle homogenates, and demonstrated to be the non-soluble, vesicle-located, ATP-Mg2+ activated sarcoendoplasmic reticulum Ca2+ pump (SERCA)  , closed 2+ and the activation of the contractile machinery, and also participated in the “relaxing factor” work, delineated the basic ECC sequence pretty much as we know it now: “The processes which would bring the contractile elements to the active state may be listed as follows: Action potential and its inward spread through the T-system … the sarcoplasmic reticulum is the site of linkage between excitation and contraction, i.e., Ca ion associated with a certain part of the sarcoplasmic reticulum is released by the influence of the electrical current field, induced by depolarization of the surface membrane, and Ca ion thus released subsequently activates the contractile system”, then, the “sarcoplasmic reticulum, exerts its relaxing effect by removing Ca ion from the contractile system in vivo”. Saul Winegrad complemented: “it is likely that … the longitudinal tubules and the intermediate cisternae … contain the calcium-sequestering system that is believed to operate during relaxation. The calcium taken up by these structures presumably then moves more slowly to the terminal cisternae, the main storage site in the muscle which has completely recovered from mechanical activity”  initiation and propagation of an action potential (AP) along the plasma membrane, 2) inward, radial spread of the depolarization along the tubular system, 3) dihydropyridine receptors (DHPR)-mediated sensing of changes in the membrane potential, 4) allosteric interaction of the DHPR with the SR Ca2+ release channels , 5) rapid release of Ca2+ from the triadic regions of the SR and transient increase of Ca2+ concentration ([Ca2+]) in the myoplasm, 6) transient activation of the contractile apparatus and the myoplasmic Ca2+ buffering system, 7) activation of the membranous-linked Ca2+ buffering and transporting system, which comprises the SERCA and the Na+/Ca2+ exchanger (NCX), and 8) appearance of the cascade of secondary mitochondria-restricted and tubular-restricted Ca2+ transients, the latter reflecting the recycling of Ca2+ through the store-operated Ca2+ entry (SOCE) mechanism.The ECC mechanism in skeletal muscle depicts a fast communication between electrical events taking place in the sarcolemma and the muscle contraction, through a cascade of global and locally restricted Ca+ rapidly increases, bringing about an AP. In most experiments, performed between 15 and 30°C, the AP depolarizes the fibre and then slightly polarizes it to positive values between +25 and +35 mV, which seems to be a safety factor for a successful AP conduction and SR Ca2+ release  or transversally  oriented membranous structures (80–100 × 35–40 nm wide), which conduct the AP at about 1 cm/s in a Native way . The so-tive way . This sttive way , a cruci2+ from the SR. The central actors in this process are the DHPR anchored to the TT and the RyR1 anchored to the SR membrane, which constitute the Ca2+ release units (CRU). DHPR  are heteropentamers formed by subunits α1 (transmembrane), γ (transmembrane), β1 (intracellular), α2 (extracellular), and δ1 (extracellular), whose function is regulated by the membrane potential. The cryoelectron microscopy (cryo-EM) reconstructions at 2.7–3.6 Å confirmed that the α1 subunit of the channel has the typical 6 × 4 structure of many voltage-gated channels, i.e., four homologous domains (DI-IV), each with six transmembrane helices , the selectivity filter (SF), and the voltage-sensing domain (VSD). The dome is a negatively charged, progressively narrowing region mainly shaped by extracellular loops above the SF of the PD, to which it guides Ca2+. The PD is made up of the S5, S6 and P helices of each domain, and several loops stabilized by multiple disulfide bonds, which create a permeation path for Ca2+ of about 60 Å in length. The SF is a specialized, narrow region, predominately formed by negatively charged aminoacids: E292 and G293 of DI, E614 and D615 of DII, E1014 and G1015 of DIII, and E1323 and A1324 of DIV. Since N617 of DII seems also to be particularly important for Ca2+ permeation , which decode the information of the tubular excitation and translates it into a signal for the RyR1. The voltage sensing function depends on the S4 enrichment in the positively charged aminoacids arginine and lysine. Their movement during the VSD operation produces a small, yet measurable, voltage-dependent intramembrane charge movement, i.e., a current, which precedes the activation of the Ca2+ release from the SR  interact with the RyR, explaining early functional observations according to which the absence of the β1 subunit eliminates the ECC . Afterwards, it is crucial to have the complete, atomic resolution structure of the DHPR-RyR or DHPR-accessory proteins-RyR complexes under different conformations, to unambiguously assign the domains that mediate their interaction and shed light on the gating mechanism. Using purified complexes or native membranes would be, at least theoretically, possible approaches to address this problem.Supramolecularly, the DHPR are arranged in groups of four, called tetrads, which alternately face the highly ordered RyR1, filling the 15–25 nm TT-SR gap .2+ channels with a big cytoplasmic moiety and a transmembrane region, inserted in rows in the jSR , four-leaf clover shaped (from the sarcoplasmic view), Ca the jSR . The eff the jSR , compare the jSR , reached channel .2+ sensing and DHPR-RyR interaction, respectively. These structures seem to be the responsible for two putative modes of activation of the RyR1: mediated by the DHPR in those RyR1 coupled to tetrads, and by Ca2+ in those RyR1 not coupled to tetrads. From the nice images published  enriched in β-sheets, which conform up to 20 domains , mainly ublished , we can ublished .2+ from the SR to the myoplasm. From the peak of the AP, the peak of the release of Ca2+ is delayed by about 2–3 ms in most experiments performed between 15 and 25°C  and reactive nitrogen species, redox state, phosphorylation/dephosphorylation status, calmodulin, S100A1, FK 506 binding protein 12 , triadin (Trisk-95 and Trisk-51), junctin, homer-1, calumenin-2 and calsequestrin (CASQ)  .2+ release in myotubes likely relies on their DHPR-binding ability and their TT-jSR tethering properties, which also mediate the precise localization of CRU (Although the SR protein-27 (SRP-27) and junctophilin (JPH)-1 interact with the RyR1 , it is nn of CRU .2+ binding protein highly expressed in the lumen of the jSR , such as the positive mitochondrial Ca2+ transients, and the negative SR and tubular Ca2+ transients. Also, as soon as the Ca2+ appears in the myoplasm, several Ca2+ buffering mechanisms are activated. Troponin C (TnC), parvalbumin (PV), ATP and the Ca2+ indicators rapidly bind Ca2+. Each of these Ca2+ binding mechanisms produces its own Ca2+ transient. Subsequently, the NCX, the mitochondria and the SERCA deal with Ca2+ with slower kinetics and the SOCE machinery recycles part of the Ca2+ extruded through the NCX. Each mechanism will be further developed in the coming paragraphs.The apparition of Ca the CRU . The ave5–106 nM  to a fib5–106 nM  , particularly abundant in muscle fibres type II, in which it can reach ∼1,000 µM. Two, high affinity, EF-hand, Catime ago . The bin release . Given ire types   channels, reflecting a phasic activation of the SOCE mechanism  fibres during a single twitch, which allowed to estimate a rather large capacity of 10–18% to buffer the cytosolic Ca2+ transient  being unbound from the TnC and PV, 2) leaving the mitochondria and 3) entering the fibre via SOCE.The final mechanism responsible for restoring the sarcoplasmic resting [Cagradient . This prgradient . ATP ande the SR . Even wh2+] was reliably quantified  researchers finally obtained structures of the DHPR and RyR at near-atomic resolution, helping explain the gating of both channels and mapping several regulators; 2) the structural and functional importance of the new main triadic regulators  was acknowledged; 3) the peak myoplasmic  is reached in the myoplasm? Is this Ca2+ enough to activate contraction? What is the kinetics of the Ca2+ reuptake? These questions have been addressed for at least 5 decades, when pioneer work demonstrated the transient increase in myoplasmic Ca2+ associated to the membrane depolarization in amphibia and arthropods muscles using murexide and aequorin as Ca2+ indicators  availability of relevant biochemical information on the ECC and fibre types, 2) the outburst of fluorescent Ca2+ indicators and 3) improvements in calibration, mathematical and computational modelling.Three attainments coincided at the time at which the field focused on Ca2+ fluxes and concentrations in mammalian muscle, especially considering the existence of at least four fibre types.Here, we will focus on how researchers integrated these three attainments to finally find a solution to the challenge of putting reliable numbers to the ECC CaThe existence of muscles with different biochemical and dynamic properties was formally acknowledged long time ago . The stu2+, many researchers presented data concerning biochemical differences in the molecular machinery involved in Ca2+ release and reuptake using the dichotomic model of slow-twitch vs. fast-twitch fibres. Later, the studies were extended to the four fibre types. Details about the molecular and biochemical differences that underlie the quantitative differences in ECC among fibre types have been presented previously and we refer the readers to those papers and the literature cited therein  TnC isoforms differ between slow and fast fibres, explaining the presence of almost twofold more Ca2+ binding sites in fibres types IIA, IIX/D and IIB, compared to type I; 3) there is a continuum increase in PV content across the four fibre types such that the fastest fibres (type IIB) have up to 300 times more than the slowest fibres (type I). Fibres IIA have about tenfold more PV than type I; 4) the differential ATP content explains the about 50% higher amount of ATP Ca2+ binding sites in the fast fibres compared to slow ones; 5) SERCA different isoforms and content among fibres result in a twofold (for IIA) or up to fivefold (for IIX/D and IIB) larger maximum Ca2+ reuptake flux rate in fast fibres compared to fibres type I; 6) up to twofold higher mitochondrial volume and a larger maximum flux rate of the mitochondrial Ca2+ uniporter (MCU) explain the threefold to fourfold higher capacity of this mechanism in fibres type I compared to fibres type II; 7) NCX1 is more abundant in fibres type I, but the capacity of the NCX3, present in fibres type II, is slightly higher; 8) STIM1 is about 1.5 times more abundant in slow compared to fast fibres, however, the SOCE total capacity seems to be higher in the latter.We can summarize the most relevant information as follows: 1) there are between twofold and threefold more CRU in the fast, compared to the slow fibres, with no difference in the isoforms. There are only about 1.5 times more triadic accessory proteins  in the fast compared to the slow muscles. Fast fibres only express CASQ1, while slow fibres have both CASQ1 and CASQ2 at a 3:1 ratio, but with a total amount of CASQ, and a total Ca2+ kinetics in different fibre types. Dynamical and molecular markers have been used to identify different fibre types in those preparations (Short (FDB) and large  fibres, either in fascicles, manually isolated or enzymatically dissociated, intact or nude, have been the most used models to study Caarations .2+ in different cell types (2+ but did not render trustable measurements of peak [Ca2+] and Ca2+ kinetics (2+ and their low Ca2+Kd (below 1 µM in vitro), limiting the range of concentrations at which the dye responds before significantly buffering Ca2+ and becoming saturated. Moreover, the calibration of non-ratiometric, slow dyes is fraught with difficulties, for instance, large errors in the Fmin estimations are common, and they may induce up to a 17% error in the [Ca2+]  and Rhod-5N, typically have an in vitro Kd between 20 and 100 µM, which rise to about 700–1,000 µM in situ, i. e, in the myoplasm or in a medium resembling the myoplasm. This very low affinity in situ is the property that makes them reliably track Ca2+ kinetics in skeletal muscle, as demonstrated because these dyes resolve every single peak of a high-frequency tetanus, and their fluorescence signals superimpose the actual Ca2+ transients  stoichiometry , less than 5% of the dye is bound to Ca2+ during a twitch, thus being far from saturation , while the fibres IIX/D and IIB share the morphology type II (MT-II); 2) single twitch MT-I signals have rise times between 1.2 and 1.8 ms and decay times of up to 80 ms. They release Ca2+ at rates between 50 and 150 μM/ms, and their peak sarcoplasmic free [Ca2+] ranges from 7 to 13 µM; 3) single twitch MT-II signals typically have rise times between 1.0 and 1.3 ms, with decay times ranging from 13 to 25 ms, being ∼3–4 times narrower than the MT-I signals; 4) MT-II fibres release Ca2+ at a huge rate of 200–250 μM/ms, and their peak sarcoplasmic free [Ca2+] ranges from 15 to 30 µM. For the sake of comparison, the peak SR release rate in mammalian cardiomyocytes ranges from 2.0 to 4.2 μM/ms  the myocytes . The difre types .2+, which, instead of its shortening ability, can be considered its main specialization.The variability in the values presented above reflects the inherent variability of the skeletal muscle biochemistry and function , its pla2+ binding mechanisms. A recent comprehensive model simulated the changes in Ca2+ concentrations and fluxes through the sarcomere of the four fibre types, considering classical  and new  Ca2+ binding sites, during single and tetanic stimulation, using Mag-Fluo-4 data  binding kinetics, consistently show that the RyR1 half-activates at ∼1–5 µM and peaks between 10 and 30 µM Ca2+ at room temperature , dynamical  and biophysical  measurements and estimations.In conclusion, although a differential biochemical data suggested different Ca2+ homeostasis, signaling, metabolism and energy production in the form of ATP, for which they have been named “the powerhouse of the cell”  to the terminal cisternae opposite to the jSR . Those t+ uptake . This toAlthough previously considered isolated organelles, mitochondria can communicate among them in different cell types . In skel2+ influx activates ATP synthesis and metabolism. Retrograde given that the ATP is also used for the SERCA to reuptake Ca2+ into SR as well as for the inhibition of local SR Ca2+ release regulation. EMC would be more important in mitochondria-enriched slow- and fast-twitch oxidative muscle than in fast-twitch glycolytic muscle , it seemed difficult to explain how Ca2+ entered the mitochondria. However, as discussed   .2+ influx from the cytoplasm to the mitochondrial intermembrane space (IMS) occurs through the porine-like, voltage-dependent anion channels (VDAC) placed in the OMM : the MCU complex  and MICU2 forming dimers and the Essential MCU Regulator (EMRE)  . MICU1 ant rises . In addint rises . The MCUnt rises .2+  (2+/H+ antiporter (mNCH or LETM1 or mHCX)  , the Ca2or mHCX)  and the or mHCX) . The firor mHCX) . The Ca2x by MCU  which masynthase . Physioll matrix . Mitochopoptosis , an effeosis . Two research groups then identified in 2005 the Stromal Interacting Molecule (STIM) as a single-pass transmembrane EF-hand protein that acts as Ca2+ sensor in the endoplasmic reticulum lumen of many cells with a low affinity of ∼200–600 μM (2+ entry units (CEU). In mammals, two STIM genes, STIM1 and STIM2, and three Orai genes, ORAI1, ORAI2 and ORAI3, have been identified , and that the rate of exchange of Ca2+ with the tubules is low , increased to 40 and 17/100 μm2 respectively, probably to compensate the reduced store Ca2+ content due to the absence of CASQ. The associated increase in Ca2+ influx by SOCE observed in these fibres, seems to maintain contractile activation in response to repetitive high frequency stimulation and resistance to fatigue , which 2+ microdomains close to it regulate the activation of SOCE locally at jSR level  skeletal muscle fibres diminish SR Caced SOCE . On the ced SOCE . On the ced SOCE , confirmTriadin and junctophilins could also participate in SOCE regulation, as their knockout or knockdown present alterations in the triad structure and an increase in the TT-SR distance, which could diminish STIM1-Orai1 interaction and SOCE . Indeed,The TRPC channels subfamily consists of seven isoforms (TRPC1-7), which are expressed in skeletal muscle, though there is controversy about TRPC5-7 . TRPC1, 2+ through Orai1 channels depends on the recruitment of TRPC1 into the plasma membrane where it is activated by STIM1  is pending. Making clearer the structural organization of the SOCE core and accessory machinery would benefit from super-resolution techniques.In summary, the current renaissance of SOCE studies doubtlessly demonstrated its existence in skeletal muscle and its activation under several conditions. Preassembled complexes and rapid responses are in tune with the main muscle specialization, i.e., its very fast Caak [Ca2+] or the mak [Ca2+], puttingIncluding stimulated emission depletion (STED), structured illumination microscopy (SIM), photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM) and their modifications, super-resolution is a novel technique with enormous potential to study skeletal muscle and help solve some issues in ECC. Membrane rearrangements and clustering of proteins, under different experimental conditions, would suitably be studied with up to a 20 nm lateral resolution in fixed and living cells . HoweverMonitoring the morphology of the neuromuscular junction using SIM and STORM allowed to propose a new model in which the ACh receptor (AChR) is not located all over the postsynaptic membrane but restricted to the area surrounding the opening of junctional folds . Also, S2+ release from the SR with a safety factor, and probably supporting part of the remodeling potential of the tubular system involved in SOCE. Resolution and quality improvements for STORM images acquisition and ensemble allow to see the boundary membranes and the lumen of the TT , which in turn could be redifferentiated to almost any cell of the three germ layers . As an e+ depolarization in a way non-dependent on external Ca2+, their ECC is very immature, from structural and functional points of view (2+ transients in response to electrical or ACh stimulation (iPSC-derived skeletal myocytes obtained in 2D cultures have AP with pretty much the same kinetics as mature muscle fibres. However, although responsive to K of view . Improvemulation . Neverth2+ and ECC related variables in iPSC-derived muscle fibres.This knowledge, along with its associated technical developments, generates a model suitable for multiple applications in biomedical studies . Envisio2+ routes generated by those proteins. Assigning reliable numbers to some of those routes has also been successful. Nonetheless, as of today, two big issues await clarification: what is the exact mechanism of DHPR-RyR coupling and what are the details of the external equilibrium of Ca2+ during ECC. Quantitative approaches, emerging techniques such as super-resolution and iPSC, and finer pharmacology through more specific drugs, may help audacious researchers obtain the answers, hopefully sooner than later.We have presented the most complete picture of the ECC up to now. Seven decades of exciting research have identified a lot of proteins involved in ECC and a plenty of Ca


Phagocytosis is one of the most polarised of all cellular activities. Both the stimulus (the target for phagocytosis) and the response  are focussed at just one part of the cell. At the locus, and this locus alone, pseudopodia form a phagocytic cup around the particle, the cytoskeleton is rearranged, the plasma membrane is reorganised, and a new internal organelle, the phagosome, is formed. The effect of signals from the stimulus must, thus, both be complex and yet be restricted in space and time to enable an effective focussed response. While many aspects of phagocytosis are being uncovered, the mechanism for the restriction of signalling or the effects of signalling remains obscure. In this review, the details of the problem of restricting chemical intracellular signalling are presented, with a focus on diffusion into the cytosol and of signalling lipids along the plasma membrane. The possible ways in which simple diffusion is overcome so that the restriction of signalling and effective phagocytosis can be achieved are discussed in the light of recent advances in imaging, biophysics, and cell biochemistry which together are providing new insights into this area. Phagocytosis is the process by which a cell internalises a particle, such as a bacterium or fungal cell , by extending pseudopodia which surround the particle and form an intracellular compartment, the phagosome ,2,3. ThiThe other important question about phagocytosis relates to the mechanisms of fusion of enzyme-containing granules with the phagosomal membrane but not the plasma membrane. As the phagosomal membrane is part of the plasma membrane until the phagosome is complete, the question arises as to how “fusion” signals are localised to the phagosomal membrane. Similarly, after completion of the phagosome, the nonmitochondrial NADPH is assembled and activated only on the phagosomal membrane and not on the plasma membrane. Again, the question arises as to what these signals are and how these signals are localised to the phagosomal membrane. Although not the main focus of this review, the evidence for possible answers to these questions about the localisation of “post-phagocytic signals” is given and briefly discussed in the second part of the review.diffusion. Diffusion is, of course, the net movement of ions or molecules from a zone of higher concentration to one of lower concentration. Thus, if an intracellular signal is generated within the cell at the contact point between the phagocytic target and the plasma membrane of the phagocyte, its intracellular concentration will be higher at that point than elsewhere in the cell and diffusion of the signal molecules away from the point of contact will occur. Diffusion is, of course, the result of the random Brownian motion of individual ions or molecules, causing the initial concentration of signals to be “diluted out” as molecules move away from a source. Although random at an individual ion/molecule level, the net effect occurs in a predictable way (2/sec).An important concept in understanding the mechanism of localised intracellular signalling is able way a and the2/s) and t is the time (s) taken  taken b. The co2+ influx from the extracellular environment generating intracellular Ca2+ signalling [2+ in the medium was an absolute requirement for phagocytosis [2+ signal may not always be obligatory, and there are early reports using nonopsonised particles in which no Ca2+ signals were found [2+ signal [2+ influx is triggered, causing a large cytosolic Ca2+ signal [2+ signal accelerates the rate of completion of phagocytosis and probably accounts for the increased phagocytic rate caused by opsonisation observed in cell populations. The flattening and spreading of neutrophils onto surfaces, which may be considered as “frustrate phagocytosis”, is also accompanied by obligatory large Ca2+ signals [2+ signal can be thought of as being the permissive signal for efficient phagocytosis.Phagocytosis by opsonised particles is accompanied by Cagnalling ,9. The eocytosis . Howeverre found ,12. Inde+ signal ,14. Howe+ signal . This Ca signals ,16. The 2+ signal could be localised to the site of phagocytosis, a useful parameter is the “diffusion length” which indicates how far from the source of the signal Ca2+ ions would travel by a defined time. The diffusion length can be calculated from knowledge of the diffusion constant, D, as 2√ would thus be 2√(800 × 0.1) = 18 μm. As this is larger than the diameter of many phagocytes , it suggests that Ca2+ ions are unlikely to act as a localised phagocytic signal. However, the diffusion constant for Ca2+ (DCa) in extracted cytosol is lower and has been measured, using Ca45 at 530 μm2/s [2/s in cytosolic extract from Xenopus oocytes [2/s [2+ is about 9.2–16 μm, which is still too large to generate localised Ca2+ signals. However, in the cytosol, Ca2+ ions bind reversibly to Ca2+ buffering agents, such as proteins and other molecules. It is estimated from the association rate (“on rate” or Kon) and buffer concentration that the lifetime of a free Ca2+ ion would be in the order of 10−5s [2+ signal would be extremely localised. However, the buffer molecule is also mobile and will diffuse, carrying Ca2+ with it until the ion is released (2+ ions. The measured diffusion constant for buffered Ca2+ is higher than for free cytosolic Ca2+ and the diffusion length of the buffered Ca2+ is extended from d1 to (d1 + d2). For Ca2+ -binding proteins with the EF hand-motif , the dissociation rate (koff) is about 10 s−1, giving a “lifetime” of the Ca2+–buffer complex of 0.1 s (100 ms). The diffusion constant of the buffered Ca2+ has been measured in cytosol extract to be 13 μm2/s. After 100 ms (when the Ca2+ ion is released), the diffusion length is only about 2.3 μm [2+ ion is free for about 1 s before being taken up into immobile Ca2+ stores, the “effective range” of buffered Ca2+ is about 5 μm [2+-buffering content of about 300 μM [2+-buffering ratio of neutrophil cytosol is similarly high, about 1000-3000:1 [2+ ion in the cytosol, there are 1000-3000 bound Ca2+ ions, so the conclusion can be extrapolated to neutrophil cytoplasm. The diffusion length predicted can also be experimentally confirmed in neutrophils by osmotically lysing Ca2+-containing lysosomes to release a small localised Ca2+ signal in the cytosol. This is achieved by the use of a dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN), which is cleaved within cathepsin C-containing lysosomes and is widely used to osmotically rupture the lysosomal membrane and release its Ca2+ content into the cytosol. In neutrophils, this procedure generated “Ca2+ puffs” with diameters of about 3 μm [2+. This suggests that the localisation of Ca2+ signals during phagocytosis is at least theoretically possible in neutrophils and probably other phagocytes. However, as the influx of Ca2+ from the extracellular environment is the important Ca2+ source for phagocytosis, understanding the mechanism for generating the Ca2+ signal is crucial.As suggested earlier, in considering the question of whether this Ca−6 cm2/s . i.e., a oocytes , or in ltes [2/s . Thus, iof 10−5s , meaningreleased . It is, t 2.3 μm . Assuminout 5 μm . Althougt 300 μM , the Ca20-3000:1 ,22, meanout 3 μm , similar2+ is very small (d1), as Ca2+ ions are rapidly bound to proteins (buffers). However, the buffer also diffuses (d2), carrying the Ca2+ ion to be released at a different location. The time between the binding and release of the Ca2+ ion depends on the dissociation rate (koff or off rate), during which time the buffered Ca2+ travels the distance d2. Ca2+-binding proteins have koff rates of about 10 s−1, giving a “lifetime” of 0.1 s. Hence, the diffusion length of the buffered Ca2+ is extended from d1 to (d1 + d2). The diffusion length of free Ca2+ signalling is via IP3, which is produced by the action of a phospholipase C on phosphatidylinositol 4,5-bisphosphate (PIP2) [3, which diffuses from the plasma membrane away from the initiation site to engage IP3 receptors on Ca2+ storage sites throughout the cell. This releases Ca2+ from the Ca2+ stores, which in turn signals plasma-membrane-channel opening through a mechanism known as “store operated Ca2+ channel” (or SOCC) opening [2+, IP3 has a long effective range [2/s, and having no buffers). Thus, for cells smaller than 24 μm, IP3 will fill the cell and act as a global messenger, thus losing any directional information as to the locus of its formation at the phagocytic site (The classic initiation mechanism for Cae (PIP2) . The twoe (PIP2) . PLC β i opening ,27. Unlive range  of 24 μmtic site .2+ influx channels . Ca2+ influx from that site, with limited diffusion distances, would retain information as to its source. In contrast, (b) is the signal for Ca2+ via PLC. The generation of IP3 with its extensive diffusion would result in the opening of Ca2+ channels from remote Ca2+ storage sites in the cell, which would open store-operated Ca2+ channels (SOCC) in the plasma membrane at all loci and so lose directional information. Route (a) shows the effect of direct coupling of the stimulus to the opening of Ca2+ Channels (SOCC) open when Ca2+ is depleted from the endoplasmic reticulum may involve the Orai1 and STIM1 system [2+ at the formed phagosome–cytosol boundary [The mechanism by which store-operated Ca1 system ,29,30. T1 system ,31,32 anboundary .2+ influx mechanism which may not involve the depletion of Ca2+ stores or IP3 generation is the opening of TRPM2 channels , Ca2+-permeable channels which are highly expressed in phagocytic immune cells [+ and Ca2+ and may be involved in immune-cell behaviour including adhesion and migration [2O2) [2+-signalling pathways [2O2 signalling. The key point, however, is that since TRPM2 activation does not involve IP3 generation, there is at least a theoretical possibility that an opsonin-triggered Ca2+ signal could remain localised to within a few microns of the phagocytic contact site if TRPM2 channels were involved. A Cane cells ,35,36. Tigration . TRPM2 ion [2O2) . There ipathways ,40,41. Apathways . As oxid2+ reporter, Quin2, reported that Ca2+ signalling was highest in the pseudopodia and periphagosomal cytoplasm. Although fura2 was reported to give a similar result [2+ during pseudopodia formation and the onset of phagocytosis cannot generally be confirmed using modern dual-wavelength or confocal imaging of a number of probes. The reported Quin2-elevated fluorescent signals in pseudopodia  were probably the result of an optical effect whereby fluors are more efficiently excited and emission-detected in the less light-scattering cytoplasm of pseudopodia [2+ signal which accompanies phagocytosis, although presumably triggered locally, causes a global elevation in cytosolic Ca2+ . In one study [2+ indicator itself affected the Ca2+ distribution by being an additional Ca2+ buffer with high mobility. The Ca2+ indicator (fluo4) was, therefore, chemically coupled to a large protein with low mobility and microinjected into neutrophils before phagocytosis. This indicator also failed to detect localised Ca2+ signalling. How, then, can Ca2+ signal the localised events that precede and trigger phagocytosis? The most likely explanation is that the protein on which Ca2+ acts has a low affinity for Ca2+, so that only protein close to the source of Ca2+ signalling will be activated, as illustrated in An early paper , using ar result , persistudopodia . When fo+ 14,16, . In one 2+ level has a very low affinity for Ca2+, the Ca2+-activated protein calpain. There is increasing evidence that calpain activity is involved in the formation of phagocytic pseudopodia [With this in mind, it is relevant that one of the many cytosolic targets of an elevated Caudopodia . For exaudopodia ,15. The udopodia , maintaiudopodia ,48,49, audopodia . After eudopodia  or sub-dudopodia  and, conudopodia . Using cudopodia . This meudopodia . The reludopodia  and ezriudopodia . Furtherudopodia .2+-activated cytosolic signalling proteins have kds between 0.1 μM and 1 μM, calpain-1 requires a Ca2+ level of 50–100 μM for activation [2+-regulated protein, has a kd for Ca2+ of about 0.3 μM, meaning that at the resting cytosolic Ca2+ level of 0.1 μM, calmodulin is not active, but when cytosolic Ca2+ rises to 1 μM, calmodulin becomes almost fully active. In contrast, calpain-1 has a kd for Ca2+ of about 30 μM, so that calpain in the bulk cytoplasm will remain inactive during the Ca2+ rise to 1 μM. The consequence of this is that calpain can only be activated very closely to the open Ca2+ channel or in a cell location where such a high Ca2+ level may be reached. One such location is within wrinkles of the plasma membrane, structures such as microridges. Phagocytes by necessity have such structures as these provide the membrane reservoir needed for the formation of the phagosomes [2+ influx into a confined volume of cytosol, it has been estimated that the Ca2+ concentration in the cytosol within the wrinkled membrane can reach hundreds of micromolar [2+ channels allow Ca2+ to enter the small volume of cytosol within the wrinkle at a faster rate than it can diffuse into the bulk cytosol through the “mouth” of the wrinkle [2+ buffers in the wrinkle space are overwhelmed, the free Ca2+ concentration can rise to very high levels. The intra-wrinkle Ca2+ has been recently measured using a construct, called EPIC3, from a low-affinity genetic Ca2+ indictor, CEPIA3 (kd for Ca2+ ≈ 11 μM), coupled to ezrin, which locates within the wrinkles and near the plasma membrane [2+ was estimated to rise to 30–80 μM [While most Cativation . For exaagosomes ,51,52. Iagosomes ,50. Fromcromolar . This is wrinkle ,57. Oncemembrane . Upon ph30–80 μM , suffici30–80 μM ,58.2+ in initiating phagocytosis, after a closed phagosome has formed, elevated periphagosomal Ca2+ may occur. It is possible that the phagosome itself provides the localised source of Ca2+ for this effect. This periphagosomal Ca2+ signal has been reported using the dual-excitation-wavelength probe, fura 2 [2+ reporter, GCaMP3, where a long-lived halo of elevated Ca2+ was detectable lasting over 6 min after phagocytic-cup formation [2+ (Ca2+ puffs) near phagosomes have also been visualised [2+ stores near phagosomes [In contrast to the role played by Ca, fura 2 , and morormation . Reportesualised , which aagosomes .2+ ions leak into the cytosol from the phagosome are probably TRPM2. These have been shown to be on the phagosomal membrane [2O2 [2O2 , it seems likely that the channels are open and generate the elevated periphagocytic Ca2+ and periphagosomal Ca2+ hotspots observed. The roles of TRPM2 opening on the plasma membrane and on the phagosome are unclear, but since TRPM2 deletion increases susceptibility to polymicrobial sepsis without reducing phagocytosis itself [The ion channels through which Camembrane  and can ane [2O2 . Since ts itself , it woul2+ and IP3 as discussed above, there are significant localised changes to the lipid composition of the membrane that form the phagocytic cup and the phagosome. Although phosphatidylserine has been implicated [2) is also elevated in the phagocytic cup of macrophages and in phagosomes [2 is simply the result of the activity of PI3K and PLCγ, both of which use PIP2 as the substrate, the first generating PIP3 and the second generating IP3. It remains uncertain why macrophages and neutrophils differ with respect to PIP2 in the formed phagosome, but the initial rise in PIP2 may be important in the initiation of the cup-forming stage of phagocytosis, which occurs before opsonin receptors have engaged in sufficient number to trigger a Ca2+ signal. An atypical ITAM sequence in moesin has been shown to transduce phagocytic signals without receptor activation [2 accumulation in the phagocytic cup results from this “primitive” nonreceptor signalling which precedes the triggering of a Ca2+ signal for large phagocytic targets. It has also been shown that mice neutrophils which do not express moesin have a defect in adhesion and spreading on to the endothelium (a type of frustrated phagocytosis) in vivo [In addition to signals which are generated locally and diffuse into the cytosol, such as Caplicated , the phoplicated ,66 and splicated ,68,69. Pagosomes ,71. Howeagosomes . In the tivation . Unopsontivation . Perhaps in vivo , whereas3), phosphatidylinositol 3,4-bisphosphate P2) [Products of phosphatidylinositol 3-Kinase, i.e., phosphatidylinositol 3,4,5-trisphosphate P2) ,76,77, aP2) , are sim2+) in the cytosol. For example, the fluorescent carbocyanine dyes DiIC18(3) or DiIC16(3), that insert their two long hydrocarbon chains  into the lipid bilayer of the plasma membrane, are free to diffuse in pure lipid bilayers, but their diffusion constant is only 9.8 μm2/s [2/s [2 in the membrane of living cells is reported to be D = 0.8 μm2/s [2/s [3 diffuses with a lateral diffusion constant of 0.9 μm2/s [2/s, and for illustration of the problem, the approximate midpoint D value of 0.5 μm2/s will be used. The laws of diffusion outlined in the earlier section also apply to lipids. In other words, free signalling lipids in the membrane tend to diffuse AWAY from the point of its generation at the phagocytic initiation site. The lateral diffusion of lipids in the plasma membrane is slower than ions , but during the time required for phagocytosis to complete (50–100 s), the signalling lipids may diffuse up to 14 μm   which isto 14 μm . Throughto 14 μm . The three panels represent the time course of phagocytosis from pre-contact of the particle with the phagocyte to cup formation and nearing phagosome closure. The target is large, having a diameter of 3 μm and circumference of about 9 μm. When the cup has formed, a phosphatidylinositol kinase adds a phosphate to form a signalling lipid such as PIP3 or its degradation, but signalling, product PIP2. The activated kinase at the initial contact point will be less than 5 μm from the edge of the cup, and the distance travelled by the lipids is shown in the last panel. Clearly, the formation of the extending pseudopodia and enclosure of the particle within a closed phagosome must be faster than the diffusion of the lipids from their source; otherwise, the lipids would leak into the surrounding plasma membrane. In the illustration, the activation of the kinase is shown by the asterisk, labelled “PI-kinase”. This is deliberately vague so as not to specify which kinase or which signalling lipid is generated. The product is labelled PIPx, where x is the number of phosphates on the inositol ring and could be one, two, or three.2+ signalling. Thus, it is possible that the lipid-signalling message generated from the centre of the particle would spill over the edges of the cup within 13 s. However, the lipid signals remain spatially restricted to the phagocytic cup and the forming phagosome ,71,83. AFrom the above, it can be seen that lipid-signalling localisation is achieved by mechanisms other than simple diffusion. Although it is not yet established what the mechanism for the restriction of diffusion might be, several possibilities exist which are discussed below.2-dependent complexes occupy the plasma membrane [3, which also associates with areas of actin polymerisation [Perhaps the simplest solution is to consider that the signalling lipids on the inner leaflet of the plasma membrane become bound to immobile elements in the cytosol. The ERM proteins ezrin, radixin, and moesin bind to both PIP2 in the membrane and immobile f-actin in the underlying cortical cytoskeleton ,85. Thusmembrane . A similrisation ,87. HoweA second possibility for the restriction of PIP2, and perhaps other lipid signals, is that diffusion is limited to the phagocytic cup because signalling molecules are prevented from escape. Such an impedance to diffusion could be a “fence” of plasma-membrane-associated proteins at the mouth of the forming phagosome which prevents PIP2 and PIP3 escape . Alterna2 is compartmentalised into plasma-membrane-ordered/raft domains [There is also the possibility that diffusion is limited by lipid domains in the plasma membrane itself. Cholesterol-enriched rafts might restrict the free diffusion of PIP2 such that the metabolism of PIP domains ,91. Sinc domains .3-specific 5-phosphatase enzymes SHIP1 and SHIP2 generating PIP2 in the plasma membrane or phagosome which is then destroyed by PTEN. The half-life of PIP3 has been estimated as less than 10 s [The third possibility is that diffusion is restricted by the degradation of the signalling lipid . PI for an The signalling lipid PIPX is here shown with three phosphates (red spheres), i.e., PIP3. If unimpeded, the signalling lipid would freely diffuse for the diffusion length, d1. However, when it encounters a phosphatase, phosphates are removed P3 becomes PIP) and the diffusion length of PIP3 is reduced to d2 by the encounter.A next possibility is one that is not often discussed, but whereas the smooth membrane surrounding the phagosome provides an ideal diffusion platform, the wrinkled and undulating plasma membrane forces molecules to travel a longer diffusion pathlength (in the z direction) for the same apparent distance from the phagosome (in the xy plane). The effect can be demonstrated using DiI, the membrane fluorescent probe, and has been used to measure the wrinkledness of the membrane during phagocytosis . HoweverThe final possibility is that either (i) each of the previously discussed possibilities, while not individually able to provide a full explanation, may together cause the effect observed; or (ii) another phenomenon will be discovered which explains the effect.phox with high affinity [Once phagocytosis is complete, the lipids on the phagosomal membrane have an important role in directing subsequent events. This is not the main subject of the review which has focussed on the signalling of phagocytosis. However, PI(3)P accumulates on the completed phagosomal membrane  and has affinity ,94,95. Iaffinity , suggestThis review has attempted to give insight into an important aspect required for a full understanding of phagocytosis. The biophysics of diffusion is presented as it relates to intracellular signalling, and provides a backdrop for interpreting some classic and more novel studies. As a result, some new ideas are presented alongside some of the well-established and older hypotheses. At the time of writing (the end of 2022), some exciting new techniques are being focussed on phagocytosis which will hopefully yield further novel insights. Phagocytosis is, of course, an ancient cell phenomenon, undertaken by single-celled animals and by single cells in higher animals. While evolution has added layer after layer of complexity, at its heart, phagocytosis must have a simplicity that could be exploited by primitive cells with primitive cell biology. Finding that “heart” of phagocytosis may open a deeper understanding of the phenomenon. It is hoped that this problem (and others) will inspire the next generation of cell biologists to be even more creative and use even more imaginative approaches to solve this, one of life’s many mysteries.


January displayed the highest incidence rate of AML, with a minimum average difference of 7% when compared to February  and a maximum average difference of 16% compared to November  and August . Such seasonal effect was consistent among subgroups according to sex and age. Our finding that AML diagnosis is seasonal strongly implies that seasonal factors, such as infectious agents or environmental triggers, influence the development and/or proliferation of disease, pointing to prevention opportunities.Until now, the role that seasonal factors play in the aetiology of acute myeloid leukaemia (AML) has been unclear. Demonstration of seasonality in AML diagnosis would provide supportive evidence of an underlying seasonal aetiology. To investigate the potential seasonal and long‐term trends in AML diagnosis in an overall population and in subgroups according to sex and age, we used population‐based data from a Spanish hospital discharge registry. We conducted a larger study than any to date of 26 472 cases of AML diagnosed in Spain between 2004 and 2015. Using multivariable Poisson generalized linear autoregressive moving average modelling, we found an upward long‐term trend, with monthly incidence rates of AML annually increasing by 0.4% [95% confidence interval (CI), 0.2%–0.6%;  Acute myeloid leukaemia (AML) is a rare disease, yet responsible for a large number of cancer‐related deaths.Mutations that cause AML can occur due to an inherited mutant gene or exposition to certain carcinogens, such as chemotherapy, radiotherapy, ionizing radiation, tobacco and benzene.Demonstration of seasonal variation in the occurrence of AML would, firstly, provide supportive evidence of aetiology by seasonal factors, such as infectious agents or environmental factors; and, secondly, focus research onto the etiological role of such factors. The value of this approach has been recognized previously, although several early studies explored and failed to draw conclusive evidence of the potential seasonality of AML.Due to a lack of consensus on the possible seasonal variation in AML incidence and the progress made within the last decade in data recording and in the development of statistical models for analysing seasonal incidence data,https://clinicaltrials.gov (Identifier: NCT03433521).The study was approved by the Ethics Committee for Clinical Research of Hospital Universitario Fundación Jiménez Díaz (EO 100/2017_FJD‐HRJC), in accordance with the Declaration of Helsinki. The study was registered on The census of the Spanish general population was obtained from the Spanish National Statistics Institute for the period 2004–2015.Conjunto Mínimo Básico de Datos (CMBD) is a national hospital discharge database that contains anonymized data for approximately 98.0% of public hospital admissions in Spain, and it covers 99.5% of the Spanish population.The N = 3471) or that only had the first decimal place of the ICD‐9 code recorded (N = 16), were excluded.Data were obtained from the CMBD on every recorded case of AML  in Spain during the period 1997–2015. We extracted information from the register of each case about the date of admission, discharge date, anonymous identifier for each patient, ICD‐9 codes, sex and date of birth from which we derived age groups as described for the at‐risk population. ICD‐9 codes included a second decimal place indicating whether the cancer was active (.00), in remission (.01), or was considered a recurrence (.02). For patients hospitalized on more than one occasion, only the record corresponding to their first diagnosis of AML was selected. To reduce the likelihood of including patients who had been diagnosed with AML before 1997, only patients admitted to hospital and diagnosed with AML in or after 2004 were included in the analysis. Patients whose first recorded diagnosis of AML was classified as remission or recurrence  method.trend (modelled as a linear trend), monthly seasonality  and December 2015 (dummy variable with two levels). The rate of AML in December 2015 was identified as an outlier from the STL analysis. To account for such an outlier in the data and remove its effect, the variable December 2015 was tested in each model. Multivariable models underwent manual backward step‐down selection to minimize the Akaike information criterion. The selection process of the appropriate lags for the autoregressive (AR) and moving average (MA) components is described in the Appendix p < 0.05.Nine separate Poisson generalized linear autoregressive moving average (GLARMA) models were fitted to evaluate the temporal dynamics in AML incidence using data for all cases, and data for each sex and age group. A detailed description of GLARMA models can be found elsewhere,A total of 26 475 individual patients with a first diagnosis of active AML were hospitalized in Spain and registered at the CMBD during 2004–2015. Information on the sex of the patient was missing for three of the cases, leaving a total of 26 472 patients in the study population. The census of the Spanish population and stratified by sex and age for years 2004–2015 can be found as Table Descriptive characteristics of sex and age of the patients are shown in Table The mean standardized yearly and monthly incidence rates of AML diagnoses over the study period were 47.62 cases per million person‐years (standard deviation: 1.01 cases per million person‐years) and 3.97 cases per million person‐months (standard deviation: 0.43 cases per million person‐months) respectively. A higher monthly incidence rate was observed in January in the overall population and in stratified analyses of sex and the age groups 5–19, 20–49, and 50–64 years , given that the other covariates are held constant. January displayed the highest incidence rate of AML, with a minimum average difference of 7% when compared to February  and a maximum average difference of 16% compared to November  and August . The incidence rate of AML for December 2015 was 0.43  times the average incidence rate for the rest of the study period. Further results from the final Poisson GLARMA models for each sex can be seen in Table A graphical representation of the fitted values from each of the nine final Poisson GLARMA models along with the actual number of cases over time is depicted in Figure Until now, the role that seasonal factors play in the aetiology of AML has been unclear.The observed age/sex‐standardized monthly incidence rates of AML in Spain over the study period were in line with the age‐standardized monthly incidence rates found in a comparable study in the United States.N = 26 472 AML cases) and that by Calip et al.N = 21 570) which analysed, respectively, the largest series of AML cases to date in Europe and the United States, provide strong evidence of a monthly variation in AML diagnosis and suggest a seasonal peak in winter months; especially in January, when the peak months found in both studies overlapped.This study showed a clear seasonal pattern in the diagnosis of AML, with the highest incidence rates of diagnosis observed in January. These findings are broadly similar to those in a large US‐based study by Calip et al. who found a peak of adult AML diagnoses during December–January from 1992 to 2008.In contrast, however, earlier studies using cancer registry data from the United States and Europe have failed to find definitive evidence of seasonality in AML diagnosis.In stratified analysis, we observed the presence of seasonal variation in the incidence of AML among females and males with a peak in January. This is broadly consistent with previous studies elsewhere.This study found strong evidence of seasonal variation in the diagnosis of AML, which suggests that seasonal risk factors, such as infectious agents or environmental factors, influence the development and/or proliferation of AML. Our results exhibited a significant annual peak in January. While the underlying reason for this result is unknown, it is possible that the observed peak in monthly AML risk coincides with seasonal elevations in the rates of infectious disease processes which are capable of precipitating or accelerating the course of AML. Indeed, infection has long been suspected as a possible factor in the aetiology of leukaemias.It is also conceivable that the annual peak observed in January reflects an increased diagnostic activity during winter months; higher levels of healthcare demand because of winter‐related illness may lead to the diagnosis of AML. Note, however, that the incidence rates recorded in February and March were higher than the incidence rates observed in December. According to such an explanation, one expects to find higher incidence rates in December if cases that would have otherwise been diagnosed in February and March were being detected in the two months prior because of an increased diagnostic activity. Conversely, some have suggested that observed seasonal variation in certain cancers is likely to reflect the reduced diagnostic activity during vacation months.Because the data are from a population‐based registry that had a high level of completeness and accuracy throughout the study period, the main results are unlikely to have been influenced by ascertainment or recording bias. However, several potential limitations of this study need to be considered. One limitation is that AML cases were obtained from the CMBD registry as defined by ICD‐9 and no other AML classifications were available. Hence, further studies using more modern classifications of AML like the World Health Organization classificationIn conclusion, this large study provides the strongest evidence of a seasonal variation in the diagnosis of AML, with a significant peak during the month of January. Such seasonal effect was consistent among subgroups according to sex and age. These findings could be used to raise the level of AML suspicion among haematologists where a cytopenia is detected in a complete blood count during winter months, particularly in January. Taken together, previous smaller studies have provided conflicting evidence, likely due to lower power and/or to the use of less advanced statistical approaches. Most studies assessing seasonal trends in AML diagnosis do so in northern‐hemisphere countries with temperate climates. Investigation of seasonality in cohorts from a wide range of geographical areas would be informative to determine the influence of latitude and climate on AML incidence. The demonstration of seasonality in our study strongly implies that seasonal factors, such as infectious agents or environmental triggers, influence the development and/or proliferation of AML, pointing to prevention opportunities. Analyses were stratified by important person‐level covariates such as sex and age that may confound seasonal trends in diagnoses. However, future studies that include more explanatory variables at individual level such as comorbid conditions and environmental exposure would augment the current results, providing further understanding into seasonal variation of AML diagnoses and the aetiology behind the observed variation.The study was conceived and designed by Juan Manuel Alonso‐Domínguez and Fernando Sánchez‐Vizcaíno. The funding for the project leading to this publication was acquired by Juan Manuel Alonso‐Domínguez and Fernando Sánchez‐Vizcaíno. The data were acquired by Raquel Barba, Maria Dolores Martin and Juan Manuel Alonso‐Domínguez. The data curation was carried out by Fernando Sánchez‐Vizcaíno and Carmen Tamayo. The methodology, formal analysis and visualization were conducted by Fernando Sánchez‐Vizcaíno. The manuscript was drafted by Fernando Sánchez‐Vizcaíno and Juan Manuel Alonso‐Domínguez. The manuscript was revised critically for important intellectual content by Carmen Tamayo, Fernando Ramos, Daniel Láinez‐González, Juana Serrano‐López, Raquel Barba, Maria Dolores Martin and Pilar Llamas. All authors gave final approval for publication.This study was supported by research funding from Celgene S.L.Juan Manuel Alonso‐Dominguez received research funding from Incyte Corporation, Pfizer International, and Astellas Pharma Inc. outside the present work.Appendix S1Click here for additional data file.


Drosophila melanogaster, phlpp, resulted in defective spindle orientation in Drosophila neuroblasts. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) is the main channel regulating fluid transport in this system, stimulated by phosphorylation by protein kinase A and inhibited by the AMP-activated protein kinase AMPK. During lumen expansion, CFTR remains open through the action of PHLPP1, which stops activated AMPK from inhibiting ion transport through CFTR. In the absence of PHLPP1, the restraint on AMPK activity is lost and this tips the balance in the favour of channel closing, resulting in the lack of lumen expansion and accumulation of mucus.Complex organ development depends on single lumen formation and its expansion during tubulogenesis. This can be achieved by correct mitotic spindle orientation during cell division, combined with luminal fluid filling that generates hydrostatic pressure. Using a human 3D cell culture model, we have identified two regulators of these processes. We find that pleckstrin homology leucine-rich repeat protein phosphatase (PHLPP) 2 regulates mitotic spindle orientation, and thereby midbody positioning and maintenance of a single lumen. Silencing the sole PHLPP family phosphatase in  Summary: A human 3D culture model reveals that pleckstrin homology leucine-rich repeat phosphatase 1 is a previously unidentified regulator of the cystic fibrosis transmembrane conductance regulator channel. Epithelial tubulogenesis typically forms a single lumen surrounded by a polarised epithelium. For several organs like the colon, this relies on fluid secretion secondary to apical ion transport, which results in lumen expansion. The chloride and bicarbonate channel cystic fibrosis transmembrane conductance regulator (CFTR) is the main driver of fluid transport in colon tubulogenesis . MutatioCFTR contains two membrane-spanning domains, two nucleotide-binding domains and a central regulatory (R) domain. Activity requires phosphorylation by cAMP-activated protein kinase A (PKA) at consensus sites in the R domain , resultiin vivo. This model presents the advantage of being able to study epithelial morphogenesis in the context of intestinal development in vitro. In this system, lumen position is defined during the first cell division. The apical membrane initiation site (AMIS) is established between the two dividing cells, at the site of the midbody. The midbody forms in the cleavage furrow as a last step during cytokinesis and has been shown to act as a polarity signal, determining whether the membrane develops an apical or basal character, and also directing the site of trafficking for further expansion of the central lumen  pathway . AKT is In this study, we show that PHLPP1 and PHLPP2 play different roles in the context of lumen development. Using a human intestinal 3D model, we find that PHLPP2 regulates mitotic spindle orientation and thereby midbody positioning and maintenance of a single lumen. Importantly, CFTR is the main channel regulating fluid transport in this system, and we find that CFTR activity is inhibited in the absence of PHLPP1. During lumen expansion, CFTR remains open through the action of PHLPP1, which stops activated AMPK from inhibiting ion transport through CFTR. In the absence of PHLPP1, the restraint on AMPK activity is lost and this tips the balance in the favour of channel closing, resulting in the lack of lumen expansion.Fig. S1A). The lack of lumen expansion was distinct from defects observed upon PHLPP2 depletion that instead caused the formation of multiple lumens . aPKC localisation remained unchanged, while E-cadherin was no longer exclusively at the plasma membrane upon PHLPP1 depletion . Importantly, depleting Caco-2 cells of both PHLPP1 and PHLPP2 gave rise to an additional category consisting of multiple lumens that were unable to expand .In an attempt to determine how PHLPP phosphatases regulate lumenogenesis, we first tested the effects of depleting PHLPP1 and PHLPP2 expression in Caco-2 colorectal adenocarcinoma cells A,B. Theso expand E,F. Thisin vivo in Drosophila melanogaster larval neuroblasts, an established model to study mitotic spindle orientation , validating the Caco-2 cell line as a model for CFTR-driven luminal filling. cAMP stimulates PKA-dependent fluid filling by activation of CFTR , suggesting that PHLPP1 regulates CFTR activity. Importantly, we could show that CFTR localisation remained apical upon PHLPP1 depletion, confirming that a trafficking defect is not responsible for the lumen expansion defect observed .As CFTR is the main ion channel in the colon, where it regulates fluid filling , we firs of CFTR . IncubatFig. S3C), suggesting that increased AMPK activity inhibits lumen expansion.AMPK has been found to be dephosphorylated at Thr172 by PHLPP1 . We therFig. S3D-F). This suggests that AMPK hyperactivation when PHLPP1 is depleted is the cause of CFTR filling defects. To test this, we made stable cell lines expressing wild-type CFTR or CFTR S768A, which cannot be phosphorylated by AMPK . We therefore conclude that PHLPP1 facilitates lumen expansion through AMPK, thereby inhibiting the ability of AMPK to phosphorylate CFTR and restrict luminal filling.As PHLPP1 depletion phenocopied CFTR inhibition and AMPK activation, we hypothesised that PHLPP1 might regulate CFTR through AMPK . In Caco by AMPK . ExpressN-[acetyl]-l-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester DAPT as well as semi-wet interface combined with mechanical stimulation  was used for western blotting . PHLPP2 2 supplemented with 15% FCS, 100 U ml−1 penicillin and 1 μg ml−1 streptomycin (Gibco). For siRNA studies, 8×105 cells were seeded into 6 cm dishes 1 day before transfection. Transfection was performed using Lipofectamine RNAi Max (Invitrogen) according to the manufacturer's specifications. One day post-transfection, cells were embedded in matrigel and collagen for a period of 5 days, as previously described (Caco-2 cells were purchased from American Type Culture Collection (ATCC) and authenticated on 12.03.2019 at 100% match by ATCC. Cell lines were routinely tested for mycoplasma contamination. Caco-2 cells were routinely cultured in DMEM at 37°C and 5% COescribed , unless 2HPO4, 35 mM NaH2PO4, 75 mM NaN3, 0.5% BSA, 2% Triton X-100, 0.4% Tween-20). Transwells were permeabilised in 0.3% Triton X-100 for 30 min and blocked in 5% BSA containing 15% FCS for 1 h. Next, cysts and transwells were incubated with primary antibodies diluted in 3% BSA in PBS overnight, followed by a 2 h secondary antibody incubation, and counterstained with the DNA dye Hoechst 33342 and Alexa Fluor 488/647 phalloidin. Confocal images were acquired on a Zeiss LSM 710, 780 or 880 confocal laser-scanning microscope. At least ten images were captured for each representative image depicted in the figures, and a minimum of three experiments was performed (n indicated in each figure).Cysts and transwells were fixed with 4% methanol-free formaldehyde for 60 min at room temperature. Cysts were washed and permeabilised by three 20 min washes in wash buffer . Lentiviral particles were packaged using a third generation packaging system . Caco-2 Mitotic spindle angles were measured by making a line at the apical membrane , a helper line  and a line through the centrosomes (spindle axis) in metaphase cells. The angle between the helper line and mitotic spindle axis was measured using Image J. Relative spindle angles, defined as the difference between the theoretical angle (90°) and the measured mitotic spindle angle, was calculated.1v1 ;attP2y, 36303) and phlpp RNAi#1 flies (P{TRiP.HMC04703}attP40) were from Bloomington Drosophila Stock Centre , whereas phlpp RNAi#2  and phlpp RNAi#3  were obtained from Vienna Drosophila Resource Centre . Knockdown in NBs was performed by crossing virgins of the NB driver line UASDicer-2;insc-Gal4, UAS-CD8::GFP  acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) treatment combined with a semi-wet interface and mechanical stimulation as described by others (Enterocytes were differentiated into mucus-producing cells using y others . BrieflyP-values when comparing lumen phenotypes resulting from knockdown treatment to controls. One-way analysis of variance was used to obtain P-values when comparing lumen phenotypes resulting from pharmacological treatment to control. The statistical tests performed for each experiment are specified in the figure legends. P-values are indicated for each experiment.Two-way analysis of variance was used to obtain


Here we examined the issue, focusing on the interaction of NCLX with an SR Ca2+ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX–SERCA2, than for NCLX–ryanodine receptor and NCLX–Na+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.The mitochondrial Na An insufficient mitochondrial Ca2+ may disable the adequate energy production by the mitochondria, whereas an excess of mitochondrial Ca2+ opens mitochondrial permeability transition pores, causing cell death .2+ in cardiomyocytes is balanced by an influx via the Ca2+ uniport (CUmit) activity and by an efflux via the Na+-Ca2+ exchange (NCXmit) or the H+-Ca2+ exchange (HCXmit) activities with the former plays the major part [mit, NCLX for NCXmit, and possibly Letm1 for HCXmit—and the physiological/pathophysiological roles of these proteins in the heart have been extensively studied [The mitochondrial Cajor part ,4 . It studied ,10,11,122+ signaling [mit activity was higher at the IFN than at the PNM, while the Ca2+ efflux activity was comparable between the two [+ channel Nav1.5, which was prominent at the SSM but not at the IFM [2+ dynamics was also suggested. De La Fuente et al. [mit were higher at mitochondria facing the junctional sarcoplasmic reticulum (jSR), where the sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor (RyR) 2 exists, than at the Percoll-purified mitochondria, whereas the protein expression of NCLX and activity of NCXmit were higher at the Percoll-purified mitochondria than those at the jSR-attached mitochondria. Interestingly, our group found that an NCLX reduction decreased the SR/endoplasmic reticulum (ER) Ca2+ content, which might be attributable to the diminished activity of the SR/ER Ca2+ pump (SERCA) in HL-1 cardiomyocytes, in murine sinoatrial node cells, as well as in B lymphocytes [2+ signaling; however, there is no information to date on the spatial relationships of NCLX with SERCA and on their physiological impact. In the present study, we explored the regional distribution of NCLX, especially focusing on the coupling with SERCA, in cardiomyocytes. We uncovered that NCLX and SERCA were localized in close proximity to each other, and that the biased localization of mitochondrial and SR Ca2+ channels/transporters contributes to efficient Ca2+ cycling between the mitochondria and SR in cardiomyocytes.In ventricular myocytes, the mitochondria are densely and regularly arranged. According to the spatial distributions, they are divided into three groups—subsarcolemmal mitochondria (SSM) just beneath the sarcolemma, interfibrillar mitochondria (IFM) between the myofibrils, and perinuclear mitochondria (PNM) near the nucleus—and the heterogeneous distribution may contribute to dealing with region-specific cellular functions, such as region-specific energy demands and Caignaling ,14. A re the two . Another the IFM . In addie et al. ,18 reporphocytes ,20,21. T+-Ca2+ exchanger (NCX) 1, in which both the N- and C-terminus are facing extracellular space, confirming that no positive signals were obtained in any pairs of the NCLX–NCX1 combination  and review [First, we studied the molecular interactions of exogenously expressed NCLX with four isoforms of SERCA—SERCA1, SERCA2A, SERCA2B, and SERCA3—in HEK293 cells. SERCA1 and SERCA2A are expressed predominantly in fast-twitch skeletal muscle and cardiac muscle, respectively, whereas SERCA2B and SERCA3 are ubiquitously expressed in broad types of tissues (see review ). To tesbination B,C. It sbination C. Regardd review ). From tmit activity was high in excitable tissues such as the heart [mit activity was low in non-excitable tissues such as the liver and kidney [Western blot analyses of isolated mitochondria using our custom-made anti-NCLX antibody  showed ahe heart ,26,27,28d kidney .+/K+ ATPase α-1 subunit were negligible in the preparations  . On the other hand, a considerable portion of the NCLX-derived signals, both on the stripes and in-between the stripes, appeared close to the SERCA2-derived signals. Co-localization analyses of the region revealed that the Pearson’s coefficient was the highest for the NCLX–SERCA2 images  . Consist2 images D. In add2 images E. Taken 2+ dynamics of cardiomyocytes using a HL-1 cell line. HL-1 has high amounts of mitochondria and SR, which are intricately tangled with each other as in adult ventricular myocytes, while the T-tubules are less developed [2+ dynamics in intact cells by expressing the SR/ER-specific Ca2+ sensor protein Cameleon D1ER, which was based on a ratiometric fluorescence resonance energy transfer (FRET) [2+ content and showed a slower reuptake of Ca2+ into emptied SR [2+ handlings caused by long-term NCLX reductions using siRNA, such as those caused by a physical distortion between mitochondria and the SR/ER as found in B lymphocytes [50 = 0.36 μM [2+ level—before an application of 10 mM of caffeine was significantly smaller, while that ratio in the presence of caffeine was comparable, in the cells treated with 5 μM CGP-37157  . We founphocytes , we phar 0.36 μM )  and NmSC were removed, also generated action potentials and cytosolic Ca2+ transients similar to the MSI model  and NCXmit facing cytoplasm (NCXmit_cyt) (non-MSI model with high CUmit_cyt and NCXmit_cyt) was constructed. The model failed to reproduce the experimental data on the effects of NCLX inhibition, indicating important roles of the biased distribution of CUmit and NCXmit  on the SR/ER, the voltage dependent anion channels (VDACs) on the outer membrane of mitochondria, and the CUmit on the inner membrane of mitochondria, have been well studied , are crucial to Ca reviews ,38,39,40affected . De La Faffected  reportedcoupling . They al and jSR . Here, w and jSR . Moreove removal . It shoumit attenuation induced a reduction in the SR Ca2+ content and firing rate [2+ accumulation in the mitochondria—i.e., the Ca2+ buffering effect of mitochondria. The mitochondrial Ca2+ buffering might have been overestimated in the previous model study, because we found that the simulation of a caffeine application protocol using the previous model resulted in a large increase in mitochondrial Ca2+ to an unphysiological level. In the present study, we simply assumed that high mitochondrial Ca2+ attenuates CUmit to suppress an unphysiological mitochondrial Ca2+ overload during a caffeine application protocol. More quantitative experimental data, such as on mitochondrial Ca2+–ΔΨ depolarization–CUmit activity relationships, are required for constructing a detailed mitochondrial model. In addition, another Ca2+ extruding system, HCXmit, was not considered in this mathematical model because the contribution of HCXmit in the cardiomyocytes is believed to be small [mit, it may play a significant role. To overcome the above limitation, further experimental and modelling studies remain to be performed.In our previous simulation study using a HL-1 cell model, which did not consider NCLX–SERCA coupling, a NCXing rate . The redbe small ,42; howe+-dependent, and CGP-37157-dependent Ca2+ efflux activity in the 45Ca2+ retention assay [There is a deviation between our findings and those by De La Fuente et al. —the NCLXon assay . At preson assay  might haon assay , they mion assay . We cannon assay  would clhttps://harrier.nagahama-i-bio.ac.jp/sosui/mobile/ (accessed on 17 December 2021) [The topology of NCLX has not been clarified yet. In Kostic et al. , NCLX waer 2021) ). On theer 2021)  to posseer 2021)  and Koster 2021) . A recener 2021) , showed er 2021) . The res2+ transfer from the mitochondria to the SR/ER should also be operative in other types of cells. In fact, we previously found the modulation of ER Ca2+ handlings via NCLX in B lymphocytes, where SERCA2 and SERCA3 were expressed, contributing to the cytosolic Ca2+ response to an antigen receptor stimulation and chemotaxis [2+ transfer in these cells. Recent findings suggested a Ca2+ dysregulation in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease . The reviews ,51). Int reviews ,53,54. IThe HEK293 cell line (ATCC CRL-1573) was purchased from Summit Pharmaceuticals International and was maintained according to the manufacture’s protocol. The mouse open reading frame cDNA clones in pCMV6-Entry vector coding for SERCA1 (NM_007504), SERCA2A (NM_009722), SERCA2B (NM_001110140), and SERCA3 (NM_016745) were purchased from Origene Technologies. The mouse open reading frame cDNA clone coding for NCX1 (NM_011406) was a kind gift from Prof. Ottolia. The bimolecular fluorescence complementation assay was performed using a CoralHue Fluo-chase Kit (MBL), according to the manufacture’s protocol. In brief, the PCR-amplified cDNA fragments of mouse NCLX (NM_133221.2) , four SEAll animal experimental procedures were approved by the Animal Research Committee, University of Fukui. Male 8–14 weeks old C57BL/6J mice , which were housed in a 12 h light–dark cycle with ad libitum access to food and water, were used for the experiments.The mice were heparinized  and sacrificed by cervical dislocation. The liver, kidney and left ventricle were quickly excised after thoracotomy. Mitochondria were isolated by a differential centrifugation method and Western blot analyses were performed as described previously ,27. The + glutamate, 30 mM KCl, 10 mM KH2PO4, 1 mM MgCl2, 20 mM taurine, 0.3 mM EGTA, 10 mM glucose, and 10 mM HEPES (pH7.2 with KOH). Immunofluorescence analyses of isolated cardiac mitochondria and isolated ventricular myocytes which were pre-loaded with or without 1 μM MitoTracker Orange CMTMRos  were performed as described previously [+/K+ ATPase α-1 subunit . The secondary antibodies used for the confocal microscopic analyses were: anti-rabbit IgG Alexa Fluor 488  for the NCLX and MCU, and anti-mouse IgG Alexa Fluor 546  or anti-mouse IgG Alexa Fluor 633  for the SERCA2, RyR, and Na+/K+ ATPase α-1 subunit. The secondary antibodies used for the super-resolution microscopic analyses were: anti-rabbit IgG CF568  for the NCLX and anti-mouse IgG Alexa Fluor 647  for the SERCA2, RyR, and Na+/K+ ATPase α-1 subunit.Left ventricular myocytes were isolated by treating the adult mouse heart with collagenase using a Langendorff-type coronary perfusion , and stoeviously . The prieviously ), anti-MFor the analyses of isolated cardiac mitochondria, the images were acquired using a laser scanning confocal microscope (Olympus FV1200) with a ×100 oil objective lens (NA 1.35). The excitation/emission wavelengths were 473/485–545 nm, 559/570–625 nm, and 635/655–755 nm for the Alexa Fluor 488, MitoTracker Orange CMTMRos, and Alexa Fluor 633, respectively.2) was calculated.For the analyses of isolated ventricular myocytes, the images were acquired using a laser scanning confocal microscope (Leica TCS SP II) with a ×100 oil objective lens (NA1.40). The excitation/emission wavelengths were 488/502–524 nm, 543/555–651 nm, and 543/558–651 nm for the Alexa Fluor 488, MitoTracker Orange CMTMRos and Alexa Fluor 546, respectively. The quality of the custom-made anti-NCLX antibody in the immunofluorescence study was confirmed in NCLX knockdown HL-1 cells . It was +/K+ ATPase α-1 subunit, respectively. The output of the activation 405 nm laser was set to 3%, and that of the imaging lasers of 561 nm and 647 nm were set to 100%. The data were processed to reconstruct the super-resolution images using NIS-Elements software (Nikon). For the co-localization analyses of the NCLX with various proteins, the ROIs with 12 μm × 4 μm—i.e., 1500 pixels × 500 pixels—in the middle of the myocytes were analyzed using the “Just Another Co-localization Plugin (JACoP)” of Image J. The offset of each image was set automatically to avoid an arbitrary judgement, then the Pearson’s correlation coefficients [Super-resolution fluorescence imaging was performed using an N-STORM  equipped with a ×100 oil objective lens (NA 1.49) and an ORCA-Flash 4.0 digital CMOS camera . In each acquisition run, 20,000 frames and 10,000 frames of 40 μm × 40 μm were recorded for the NCLX and for the SERCA2, RyR, and Naficients  were cal2+ indicator Cameleon D1ER/pcDNA3, which was a kind gift from Dr. Tsien [2PO4, 0.5 mM MgCl2, 1.8 mM CaCl2, 5.5 mM glucose and 5 mM HEPES (pH 7.4 with NaOH). Then, 10 mM caffeine in Tyrode’s solution was applied for 1 min to empty the SR Ca2+ by switching the solution using a valve controlled gravity perfusion system . The cells were excited at 435 ± 10 nm and fluorescence image pairs at 480 ± 30 (CGP) and 535 ± 40 nm (YFP), which were separated with a W-View system (Hamamatsu Photonics), were recorded every 4 sec using a cooled CCD digital camera . The images were analyzed using the AQUACOSMOS software (Hamamatsu Photonics). The YFP/CFP fluorescence intensity ratio of each cell was considered as the SR Ca2+ level of the cell, and the ΔYFP/CFP just before and during the caffeine application was considered as the SR Ca2+ content. The recovery rate of the YFP/CFP after the removal of caffeine was considered as the SR Ca2+ reuptake rate, which was evaluated by calculating the half time (t1/2) from a minimum YFP/CFP during the caffeine application to the steady-state value at 3 min after the removal of caffeine. In order to block NCLX, the experiments were performed in the presence of 5 μM CGP-37157.The HL-1 cell line was a kind gift from Dr. Claycomb  and was r. Tsien , was trar. Tsien . In brieA mathematical model of HL-1 cardiomyocytes considering MSI was developed based on our previous model . The dett-test, paired t-test, and one-way ANOVA for unpaired two group comparisons, paired two group comparisons, and multiple comparisons, respectively (SigmaPlot 14.0). A p-value of <0.05 was determined as a statistically significant difference.The statistical analyses were performed by a Student’s