Information

9.14: Methods of Intracellular Signaling - Biology

9.14: Methods of Intracellular Signaling - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

The induction of a signaling pathway depends on the modification of a cellular component by an enzyme. The following are some of the more common events in intracellular signaling.

Observe an animation of cell signaling at this site.

Phosphorylation

One of the most common chemical modifications that occurs in signaling pathways is the addition of a phosphate group (PO4–3) to a molecule such as a protein in a process called phosphorylation. The phosphate can be added to a nucleotide such as GMP to form GDP or GTP. Phosphates are also often added to serine, threonine, and tyrosine residues of proteins, where they replace the hydroxyl group of the amino acid (Figure 1). The transfer of the phosphate is catalyzed by an enzyme called a kinase. Various kinases are named for the substrate they phosphorylate. Phosphorylation of serine and threonine residues often activates enzymes. Phosphorylation of tyrosine residues can either affect the activity of an enzyme or create a binding site that interacts with downstream components in the signaling cascade. Phosphorylation may activate or inactivate enzymes, and the reversal of phosphorylation, dephosphorylation by a phosphatase, will reverse the effect.

Second Messengers

Second messengers are small molecules that propagate a signal after it has been initiated by the binding of the signaling molecule to the receptor. These molecules help to spread a signal through the cytoplasm by altering the behavior of certain cellular proteins.

Calcium ion is a widely used second messenger. The free concentration of calcium ions (Ca2+) within a cell is very low because ion pumps in the plasma membrane continuously use adenosine-5′-triphosphate (ATP) to remove it. For signaling purposes, Ca2+ is stored in cytoplasmic vesicles, such as the endoplasmic reticulum, or accessed from outside the cell. When signaling occurs, ligand-gated calcium ion channels allow the higher levels of Ca2+ that are present outside the cell (or in intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of cytoplasmic Ca2+. The response to the increase in Ca2+ varies, depending on the cell type involved. For example, in the β-cells of the pancreas, Ca2+ signaling leads to the release of insulin, and in muscle cells, an increase in Ca2+ leads to muscle contractions.

Another second messenger utilized in many different cell types is cyclic AMP (cAMP). Cyclic AMP is synthesized by the enzyme adenylyl cyclase from ATP (Figure 2). The main role of cAMP in cells is to bind to and activate an enzyme called cAMP-dependent kinase (A-kinase). A-kinase regulates many vital metabolic pathways: It phosphorylates serine and threonine residues of its target proteins, activating them in the process. A-kinase is found in many different types of cells, and the target proteins in each kind of cell are different. Differences give rise to the variation of the responses to cAMP in different cells.

Present in small concentrations in the plasma membrane, inositol phospholipids are lipids that can also be converted into second messengers. Because these molecules are membrane components, they are located near membrane-bound receptors and can easily interact with them. Phosphatidylinositol (PI) is the main phospholipid that plays a role in cellular signaling. Enzymes known as kinases phosphorylate PI to form PI-phosphate (PIP) and PI-bisphosphate (PIP2).

The enzyme phospholipase C cleaves PIP2 to form diacylglycerol (DAG) and inositol triphosphate (IP3) (Figure 3). These products of the cleavage of PIP2 serve as second messengers. Diacylglycerol (DAG) remains in the plasma membrane and activates protein kinase C (PKC), which then phosphorylates serine and threonine residues in its target proteins. IP3 diffuses into the cytoplasm and binds to ligand-gated calcium channels in the endoplasmic reticulum to release Ca2+ that continues the signal cascade.

Learning Objectives

Ligand binding to the receptor allows for signal transduction through the cell. The chain of events that conveys the signal through the cell is called a signaling pathway or cascade. Signaling pathways are often very complex because of the interplay between different proteins. A major component of cell signaling cascades is the phosphorylation of molecules by enzymes known as kinases. Phosphorylation adds a phosphate group to serine, threonine, and tyrosine residues in a protein, changing their shapes, and activating or inactivating the protein. Small molecules like nucleotides can also be phosphorylated. Second messengers are small, non-protein molecules that are used to transmit a signal within a cell. Some examples of second messengers are calcium ions (Ca2+), cyclic AMP (cAMP), diacylglycerol (DAG), and inositol triphosphate (IP3).


Chapter 9. Cell Communication

Figure 9.1 Have you ever become separated from a friend while in a crowd? If so, you know the challenge of searching for someone when surrounded by thousands of other people. If you and your friend have cell phones, your chances of finding each other are good. A cell phone’s ability to send and receive messages makes it an ideal communication device. (Credit: modification of work by Vincent and Bella Productions)
  • 9.1 Signaling Molecules and Cellular Receptors
  • 9.2 Propagation of the Signal
  • 9.3 Response to the Signal

Calcium and Cell Function

Calcium and Cell Function, Volume 7 covers the signal transduction across the cell membrane. The book discusses phosphoinositides and calcium signaling calmodulin-stimulated adenylate cyclases and calcium/calmodulin-dependent protein kinases. The text also describes the regulation of gene expression by calcium the dynamics of intracellular calcium concentration during mitosis and the methods for the measurement of intracellular ionized calcium in mammalian cells. Pharmacologists, physiologists, and people involved in the study of calcium and cell function will find the book invaluable.

Calcium and Cell Function, Volume 7 covers the signal transduction across the cell membrane. The book discusses phosphoinositides and calcium signaling calmodulin-stimulated adenylate cyclases and calcium/calmodulin-dependent protein kinases. The text also describes the regulation of gene expression by calcium the dynamics of intracellular calcium concentration during mitosis and the methods for the measurement of intracellular ionized calcium in mammalian cells. Pharmacologists, physiologists, and people involved in the study of calcium and cell function will find the book invaluable.


Contents

In 1914, John S. Dexter noticed the appearance of a notch in the wings of the fruit fly Drosophila melanogaster. The alleles of the gene were identified in 1917 by American evolutionary biologist Thomas Hunt Morgan. [4] [5] Its molecular analysis and sequencing was independently undertaken in the 1980s by Spyros Artavanis-Tsakonas and Michael W. Young. [6] [7] Alleles of the two C. elegans Notch genes were identified based on developmental phenotypes: lin-12 [8] and glp-1. [9] [10] The cloning and partial sequence of lin-12 was reported at the same time as Drosophila Notch by Iva Greenwald. [11]

The Notch protein spans the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression. [12]

The cleavage model was first proposed in 1993 based on work done with Drosophila Notch and C. elegans lin-12, [13] [14] informed by the first oncogenic mutation affecting a human Notch gene. [15] Compelling evidence for this model was provided in 1998 by in vivo analysis in Drosophila by Gary Struhl [16] and in cell culture by Raphael Kopan. [17] Although this model was initially disputed, [1] the evidence in favor of the model was irrefutable by 2001. [18] [19]

The receptor is normally triggered via direct cell-to-cell contact, in which the transmembrane proteins of the cells in direct contact form the ligands that bind the notch receptor. The Notch binding allows groups of cells to organize themselves such that, if one cell expresses a given trait, this may be switched off in neighbouring cells by the intercellular notch signal. In this way, groups of cells influence one another to make large structures. Thus, lateral inhibition mechanisms are key to Notch signaling. lin-12 and Notch mediate binary cell fate decisions, and lateral inhibition involves feedback mechanisms to amplify initial differences. [18]

The Notch cascade consists of Notch and Notch ligands, as well as intracellular proteins transmitting the notch signal to the cell's nucleus. The Notch/Lin-12/Glp-1 receptor family [20] was found to be involved in the specification of cell fates during development in Drosophila and C. elegans. [21]

The intracellular domain of Notch forms a complex with CBF1 and Mastermind to activate transcription of target genes. The structure of the complex has been determined. [22] [23]

The Notch signaling pathway is important for cell-cell communication, which involves gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch signaling also has a role in the following processes:

    function and development [24][25][26][27]
  • stabilization of arterial endothelial fate and angiogenesis[28]
  • regulation of crucial cell communication events between endocardium and myocardium during both the formation of the valve primordial and ventricular development and differentiation [29]homeostasis, as well as implications in other human disorders involving the cardiovascular system [30]
  • timely cell lineage specification of both endocrine and exocrine pancreas[31]
  • influencing of binary fate decisions of cells that must choose between the secretory and absorptive lineages in the gut [32]
  • expansion of the hematopoietic stem cell compartment during bone development and participation in commitment to the osteoblastic lineage, suggesting a potential therapeutic role for notch in bone regeneration and osteoporosis [33]
  • expansion of the hemogenic endothelial cells along with signaling axis involving Hedgehog signaling and Scl[34] lineage commitment from common lymphoid precursor [35]
  • regulation of cell-fate decision in mammary glands at several distinct development stages [36]
  • possibly some non-nuclear mechanisms, such as control of the actincytoskeleton through the tyrosine kinaseAbl[37]
  • Regulation of the mitotic/meiotic decision in the C. elegans germline [9]

Notch signaling is dysregulated in many cancers, [38] and faulty notch signaling is implicated in many diseases including T-ALL (T-cell acute lymphoblastic leukemia), [39] CADASIL (Cerebral Autosomal-Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), multiple sclerosis (MS), Tetralogy of Fallot, Alagille syndrome, and many other disease states.

Inhibition of notch signaling has been shown to have anti-proliferative effects on T-cell acute lymphoblastic leukemia in cultured cells and in a mouse model. [40] [41] [42] It has also been found that Rex1 has inhibitory effects on the expression of notch in mesenchymal stem cells, preventing differentiation. [43]

Maturation of the notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex. [44] This results in a bipartite protein, composed of a large extracellular domain linked to the smaller transmembrane and intracellular domain. Binding of ligand promotes two proteolytic processing events as a result of proteolysis, the intracellular domain is liberated and can enter the nucleus to engage other DNA-binding proteins and regulate gene expression.

Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically must be adjacent to the notch expressing cell for signaling to occur. [ citation needed ] The notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins. In Drosophila melanogaster (the fruit fly), there are two ligands named Delta and Serrate. In mammals, the corresponding names are Delta-like and Jagged. In mammals there are multiple Delta-like and Jagged ligands, as well as possibly a variety of other ligands, such as F3/contactin. [37]

In the nematode C. elegans, two genes encode homologous proteins, glp-1 and lin-12. There has been at least one report that suggests that some cells can send out processes that allow signaling to occur between cells that are as much as four or five cell diameters apart. [ citation needed ]

The notch extracellular domain is composed primarily of small cystine-rich motifs called EGF-like repeats. [45]

Notch 1, for example, has 36 of these repeats. Each EGF-like repeat is composed of approximately 40 amino acids, and its structure is defined largely by six conserved cysteine residues that form three conserved disulfide bonds. Each EGF-like repeat can be modified by O-linked glycans at specific sites. [46] An O-glucose sugar may be added between the first and second conserved cysteines, and an O-fucose may be added between the second and third conserved cysteines. These sugars are added by an as-yet-unidentified O-glucosyltransferase (except for Rumi), and GDP-fucose Protein O-fucosyltransferase 1 (POFUT1), respectively. The addition of O-fucose by POFUT1 is absolutely necessary for notch function, and, without the enzyme to add O-fucose, all notch proteins fail to function properly. As yet, the manner by which the glycosylation of notch affects function is not completely understood.

The O-glucose on notch can be further elongated to a trisaccharide with the addition of two xylose sugars by xylosyltransferases, and the O-fucose can be elongated to a tetrasaccharide by the ordered addition of an N-acetylglucosamine (GlcNAc) sugar by an N-Acetylglucosaminyltransferase called Fringe, the addition of a galactose by a galactosyltransferase, and the addition of a sialic acid by a sialyltransferase. [47]

To add another level of complexity, in mammals there are three Fringe GlcNAc-transferases, named lunatic fringe, manic fringe, and radical fringe. These enzymes are responsible for something called a "fringe effect" on notch signaling. [48] If Fringe adds a GlcNAc to the O-fucose sugar then the subsequent addition of a galactose and sialic acid will occur. In the presence of this tetrasaccharide, notch signals strongly when it interacts with the Delta ligand, but has markedly inhibited signaling when interacting with the Jagged ligand. [49] The means by which this addition of sugar inhibits signaling through one ligand, and potentiates signaling through another is not clearly understood.

Once the notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called ADAM10, cleaves the notch protein just outside the membrane. [50] This releases the extracellular portion of notch (NECD), which continues to interact with the ligand. The ligand plus the notch extracellular domain is then endocytosed by the ligand-expressing cell. There may be signaling effects in the ligand-expressing cell after endocytosis this part of notch signaling is a topic of active research. [ citation needed ] After this first cleavage, an enzyme called γ-secretase (which is implicated in Alzheimer's disease) cleaves the remaining part of the notch protein just inside the inner leaflet of the cell membrane of the notch-expressing cell. This releases the intracellular domain of the notch protein (NICD), which then moves to the nucleus, where it can regulate gene expression by activating the transcription factor CSL. It was originally thought that these CSL proteins suppressed Notch target transcription. However, further research showed that, when the intracellular domain binds to the complex, it switches from a repressor to an activator of transcription. [51] Other proteins also participate in the intracellular portion of the notch signaling cascade. [52]

Notch signaling is initiated when Notch receptors on the cell surface engage ligands presented in trans on opposing cells. Despite the expansive size of the Notch extracellular domain, it has been demonstrated that EGF domains 11 and 12 are the critical determinants for interactions with Delta. [53] Additional studies have implicated regions outside of Notch EGF11-12 in ligand binding. For example, Notch EGF domain 8 plays a role in selective recognition of Serrate/Jagged [54] and EGF domains 6-15 are required for maximal signaling upon ligand stimulation. [55] A crystal structure of the interacting regions of Notch1 and Delta-like 4 (Dll4) provided a molecular-level visualization of Notch-ligand interactions, and revealed that the N-terminal MNNL (or C2) and DSL domains of ligands bind to Notch EGF domains 12 and 11, respectively. [56] The Notch1-Dll4 structure also illuminated a direct role for Notch O-linked fucose and glucose moieties in ligand recognition, and rationalized a structural mechanism for the glycan-mediated tuning of Notch signaling. [56]

The Notch signaling pathway plays an important role in cell-cell communication, and further regulates embryonic development.

Embryo polarity Edit

Notch signaling is required in the regulation of polarity. For example, mutation experiments have shown that loss of Notch signaling causes abnormal anterior-posterior polarity in somites. [57] Also, Notch signaling is required during left-right asymmetry determination in vertebrates. [58]

Early studies in the nematode model organism C. elegans indicate that Notch signaling has a major role in the induction of mesoderm and cell fate determination. [9] As mentioned previously, C. elegans has two genes that encode for partially functionally redundant Notch homologs, glp-1 and lin-12. [59] During C. elegans, GLP-1, the C. elegans Notch homolog, interacts with APX-1, the C. elegans Delta homolog. This signaling between particular blastomeres induces differentiation of cell fates and establishes the dorsal-ventral axis. [60]

Somitogenesis Edit

Notch signaling is central to somitogenesis. In 1995, Notch1 was shown to be important for coordinating the segmentation of somites in mice. [61] Further studies identified the role of Notch signaling in the segmentation clock. These studies hypothesized that the primary function of Notch signaling does not act on an individual cell, but coordinates cell clocks and keep them synchronized. This hypothesis explained the role of Notch signaling in the development of segmentation and has been supported by experiments in mice and zebrafish. [62] [63] [64] Experiments with Delta1 mutant mice that show abnormal somitogenesis with loss of anterior/posterior polarity suggest that Notch signaling is also necessary for the maintenance of somite borders. [61]

During somitogenesis, a molecular oscillator in paraxial mesoderm cells dictates the precise rate of somite formation. A clock and wavefront model has been proposed in order to spatially determine the location and boundaries between somites. This process is highly regulated as somites must have the correct size and spacing in order to avoid malformations within the axial skeleton that may potentially lead to spondylocostal dysostosis. Several key components of the Notch signaling pathway help coordinate key steps in this process. In mice, mutations in Notch1, Dll1 or Dll3, Lfng, or Hes7 result in abnormal somite formation. Similarly, in humans, the following mutations have been seen to lead to development of spondylocostal dysostosis: DLL3, LFNG, or HES7. [65]

Epidermal differentiation Edit

Notch signaling is known to occur inside ciliated, differentiating cells found in the first epidermal layers during early skin development. [66] Furthermore, it has found that presenilin-2 works in conjunction with ARF4 to regulate Notch signaling during this development. [67] However, it remains to be determined whether gamma-secretase has a direct or indirect role in modulating Notch signaling.

Early findings on Notch signaling in central nervous system (CNS) development were performed mainly in Drosophila with mutagenesis experiments. For example, the finding that an embryonic lethal phenotype in Drosophila was associated with Notch dysfunction [68] indicated that Notch mutations can lead to the failure of neural and Epidermal cell segregation in early Drosophila embryos. In the past decade, advances in mutation and knockout techniques allowed research on the Notch signaling pathway in mammalian models, especially rodents.

The Notch signaling pathway was found to be critical mainly for neural progenitor cell (NPC) maintenance and self-renewal. In recent years, other functions of the Notch pathway have also been found, including glial cell specification, [69] [70] neurites development, [71] as well as learning and memory. [72]

Neuron cell differentiation Edit

The Notch pathway is essential for maintaining NPCs in the developing brain. Activation of the pathway is sufficient to maintain NPCs in a proliferating state, whereas loss-of-function mutations in the critical components of the pathway cause precocious neuronal differentiation and NPC depletion. [25] Modulators of the Notch signal, e.g., the Numb protein are able to antagonize Notch effects, resulting in the halting of cell cycle and differentiation of NPCs. [73] [74] Conversely, the fibroblast growth factor pathway promotes Notch signaling to keep stem cells of the cerebral cortex in the proliferative state, amounting to a mechanism regulating cortical surface area growth and, potentially, gyrification. [75] [76] In this way, Notch signaling controls NPC self-renewal as well as cell fate specification.

A non-canonical branch of the Notch signaling pathway that involves the phosphorylation of STAT3 on the serine residue at amino acid position 727 and subsequent Hes3 expression increase (STAT3-Ser/Hes3 Signaling Axis) has been shown to regulate the number of NPCs in culture and in the adult rodent brain. [77]

In adult rodents and in cell culture, Notch3 promotes neuronal differentiation, having a role opposite to Notch1/2. [78] This indicates that individual Notch receptors can have divergent functions, depending on cellular context.

Neurite development Edit

In vitro studies show that Notch can influence neurite development. [71] In vivo, deletion of the Notch signaling modulator, Numb, disrupts neuronal maturation in the developing cerebellum, [79] whereas deletion of Numb disrupts axonal arborization in sensory ganglia. [80] Although the mechanism underlying this phenomenon is not clear, together these findings suggest Notch signaling might be crucial in neuronal maturation.

Gliogenesis Edit

In gliogenesis, Notch appears to have an instructive role that can directly promote the differentiation of many glial cell subtypes. [69] [70] For example, activation of Notch signaling in the retina favors the generation of Muller glia cells at the expense of neurons, whereas reduced Notch signaling induces production of ganglion cells, causing a reduction in the number of Muller glia. [25]

Adult brain function Edit

Apart from its role in development, evidence shows that Notch signaling is also involved in neuronal apoptosis, neurite retraction, and neurodegeneration of ischemic stroke in the brain [81] In addition to developmental functions, Notch proteins and ligands are expressed in cells of the adult nervous system, [82] suggesting a role in CNS plasticity throughout life. Adult mice heterozygous for mutations in either Notch1 or Cbf1 have deficits in spatial learning and memory. [72] Similar results are seen in experiments with presenilins1 and 2, which mediate the Notch intramembranous cleavage. To be specific, conditional deletion of presenilins at 3 weeks after birth in excitatory neurons causes learning and memory deficits, neuronal dysfunction, and gradual neurodegeneration. [83] Several gamma secretase inhibitors that underwent human clinical trials in Alzheimer's disease and MCI patients resulted in statistically significant worsening of cognition relative to controls, which is thought to be due to its incidental effect on Notch signalling. [84]

The Notch signaling pathway is a critical component of cardiovascular formation and morphogenesis in both development and disease. It is required for the selection of endothelial tip and stalk cells during sprouting angiogenesis. [85]

Cardiac development Edit

Notch signal pathway plays a crucial role in at least three cardiac development processes: Atrioventricular canal development, myocardial development, and cardiac outflow tract (OFT) development. [86]

Atrioventricular (AV) canal development Edit

Ventricular development Edit

Ventricular outflow tract development Edit

Angiogenesis Edit

Endothelial cells use the Notch signaling pathway to coordinate cellular behaviors during the blood vessel sprouting that occurs sprouting angiogenesis. [101] [102] [103] [104]

Activation of Notch takes place primarily in "connector" cells and cells that line patent stable blood vessels through direct interaction with the Notch ligand, Delta-like ligand 4 (Dll4), which is expressed in the endothelial tip cells. [105] VEGF signaling, which is an important factor for migration and proliferation of endothelial cells, [106] can be downregulated in cells with activated Notch signaling by lowering the levels of Vegf receptor transcript. [107] Zebrafish embryos lacking Notch signaling exhibit ectopic and persistent expression of the zebrafish ortholog of VEGF3, flt4, within all endothelial cells, while Notch activation completely represses its expression. [108]

Notch signaling may be used to control the sprouting pattern of blood vessels during angiogenesis. When cells within a patent vessel are exposed to VEGF signaling, only a restricted number of them initiate the angiogenic process. Vegf is able to induce DLL4 expression. In turn, DLL4 expressing cells down-regulate Vegf receptors in neighboring cells through activation of Notch, thereby preventing their migration into the developing sprout. Likewise, during the sprouting process itself, the migratory behavior of connector cells must be limited to retain a patent connection to the original blood vessel. [105]

During development, definitive endoderm and ectoderm differentiates into several gastrointestinal epithelial lineages, including endocrine cells. Many studies have indicated that Notch signaling has a major role in endocrine development.

Pancreatic development Edit

The formation of the pancreas from endoderm begins in early development. The expression of elements of the Notch signaling pathway have been found in the developing pancreas, suggesting that Notch signaling is important in pancreatic development. [109] [110] Evidence suggests Notch signaling regulates the progressive recruitment of endocrine cell types from a common precursor, [111] acting through two possible mechanisms. One is the "lateral inhibition", which specifies some cells for a primary fate but others for a secondary fate among cells that have the potential to adopt the same fate. Lateral inhibition is required for many types of cell fate determination. Here, it could explain the dispersed distribution of endocrine cells within pancreatic epithelium. [112] A second mechanism is "suppressive maintenance", which explains the role of Notch signaling in pancreas differentiation. Fibroblast growth factor10 is thought to be important in this activity, but the details are unclear. [113] [114]

Intestinal development Edit

The role of Notch signaling in the regulation of gut development has been indicated in several reports. Mutations in elements of the Notch signaling pathway affect the earliest intestinal cell fate decisions during zebrafish development. [115] Transcriptional analysis and gain of function experiments revealed that Notch signaling targets Hes1 in the intestine and regulates a binary cell fate decision between adsorptive and secretory cell fates. [115]

Bone development Edit

Early in vitro studies have found the Notch signaling pathway functions as down-regulator in osteoclastogenesis and osteoblastogenesis. [116] Notch1 is expressed in the mesenchymal condensation area and subsequently in the hypertrophic chondrocytes during chondrogenesis. [117] Overexpression of Notch signaling inhibits bone morphogenetic protein2-induced osteoblast differentiation. Overall, Notch signaling has a major role in the commitment of mesenchymal cells to the osteoblastic lineage and provides a possible therapeutic approach to bone regeneration. [33]

Notch is implicated in development of alveoli in the lung. [118]

Role of Notch signaling in leukemia Edit

Aberrant Notch signaling is a driver of T cell acute lymphoblastic leukemia (T-ALL) [119] and is mutated in at least 65% of all T-ALL cases. [120] Notch signaling can be activated by mutations in Notch itself, inactivating mutations in FBXW7 (a negative regulator of Notch1), or rarely by t(79)(q34q34.3) translocation. In the context of T-ALL, Notch activity cooperates with additional oncogenic lesions such as c-MYC to activate anabolic pathways such as ribosome and protein biosynthesis thereby promoting leukemia cell growth. [121]

Notch inhibitors Edit

The involvement of Notch signaling in many cancers has led to investigation of notch inhibitors (especially gamma-secretase inhibitors) as cancer treatments which are in different phases of clinical trials. [2] [122] As of 2013 [update] at least 7 notch inhibitors were in clinical trials. [123] MK-0752 has given promising results in an early clinical trial for breast cancer. [124] Preclinical studies showed beneficial effects of gamma-secretase inhibitors in endometriosis, [125] a disease characterised by increased expression of notch pathway constituents. [126] [127]

It is possible to engineer synthetic Notch receptors by replacing the extracellular receptor and intracellular transcriptional domains with other domains of choice. This allows researchers to select which ligands are detected, and which genes are upregulated in response. Using this technology, cells can report or change their behavior in response to contact with user-specified signals, facilitating new avenues of both basic and applied research into cell-cell signaling. [128] Notably, this system allows multiple synthetic pathways to be engineered into a cell in parallel. [129] [130]


Abstract

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca 2+ - and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca 2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca 2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.


Single-Cell Intracellular Proteome Product Sheet

Our proprietary & patented “Proteomic Barcoded” IsoCode Chip, captures single cells in each microchamber, and detects the full range of phosphoproteins, enabling predictive intracellular discoveries.

P-PRAS40, P-IkBα, P-NF-kβ p65, P-Met, P-p44/42 MAPK, P-S6 Ribosomal, P-Rb, P-p90RSK, P-STAT3, P-MEK1/2, P-Stat1, P-Stat5, P-eIF4E, Cleaved PARP, Alpha Tubulin

P-Akt, P-p53, P-PD1, P-LCK, P-CD3 zeta, P-Zap70, P-CCR7, P-CD28, P-41BB, P-MEK 1/2, P-P44/42 MAPK (ERK1/2), P-Jak1, P-Jak2, P-AMPK, P-PI3K, P-mTOR, P-P21, P-LAT, P-NF-kB p65, Alpha Tubulin

The breakthrough Single-Cell Intracellular Proteome Solution allows users to analyze signaling cascades of many phosphoproteins directly from each single cell, across thousands of single cells in parallel, for the first time. The leap over existing technologies like western blot, mass spectrometry, and flow cytometry, is the Single-Cell Intracellular Proteome solution’s ability to quantify and highly multiplex 15+ intracellular analytes simultaneously from each cell, and thus detect critical protein to protein interactions and signaling networks in rare cells and cell subsets.


Methods of Intracellular Signaling

Signaling pathway induction activates a sequence of enzymatic modifications that are recognized in turn by the next component downstream.

Learning Objectives

Explain how the binding of a ligand initiates signal transduction throughout a cell

Key Takeaways

Key Points

  • Phosphorylation, the addition of a phosphate group to a molecule such as a protein, is one of the most common chemical modifications that occurs in signaling pathways.
  • The activation of second messengers, small molecules that propagate a signal, is a common event after the induction of a signaling pathway.
  • Calcium ion, cyclic AMP, and inositol phospholipids are examples of widely-used second messengers.

Key Terms

  • second messenger: any substance used to transmit a signal within a cell, especially one which triggers a cascade of events by activating cellular components
  • phosphorylation: the addition of a phosphate group to a compound often catalyzed by enzymes

The induction of a signaling pathway depends on the modification of a cellular component by an enzyme. There are numerous enzymatic modifications that can occur which are recognized in turn by the next component downstream.

One of the most common chemical modifications that occurs in signaling pathways is the addition of a phosphate group (PO4 –3 ) to a molecule such as a protein in a process called phosphorylation. The phosphate can be added to a nucleotide such as GMP to form GDP or GTP. Phosphates are also often added to serine, threonine, and tyrosine residues of proteins where they replace the hydroxyl group of the amino acid. The transfer of the phosphate is catalyzed by an enzyme called a kinase. Various kinases are named for the substrate they phosphorylate. Phosphorylation of serine and threonine residues often activates enzymes. Phosphorylation of tyrosine residues can either affect the activity of an enzyme or create a binding site that interacts with downstream components in the signaling cascade. Phosphorylation may activate or inactivate enzymes the reversal of phosphorylation, dephosphorylation by a phosphatase, will reverse the effect.

Example of phosphorylation: In protein phosphorylation, a phosphate group (PO4-3 ) is added to residues of the amino acids serine, threonine, and tyrosine.

The activation of second messengers is also a common event after the induction of a signaling pathway. They are small molecules that propagate a signal after it has been initiated by the binding of the signaling molecule to the receptor. These molecules help to spread a signal through the cytoplasm by altering the behavior of certain cellular proteins.

Calcium ion is a widely-used second messenger. The free concentration of calcium ions (Ca 2+ ) within a cell is very low because ion pumps in the plasma membrane continuously use adenosine-5′-triphosphate ( ATP ) to remove it. For signaling purposes, Ca 2+ is stored in cytoplasmic vesicles, such as the endoplasmic reticulum, or accessed from outside the cell. When signaling occurs, ligand-gated calcium ion channels allow the higher levels of Ca 2+ that are present outside the cell (or in intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of cytoplasmic Ca 2+ . The response to the increase in Ca 2+ varies, depending on the cell type involved. For example, in the β-cells of the pancreas, Ca 2+ signaling leads to the release of insulin, whereas in muscle cells, an increase in Ca 2+ leads to muscle contractions.

Another second messenger utilized in many different cell types is cyclic AMP (cAMP). Cyclic AMP is synthesized by the enzyme adenylyl cyclase from ATP. The main role of cAMP in cells is to bind to and activate an enzyme called cAMP-dependent kinase (A-kinase). A-kinase regulates many vital metabolic pathways. It phosphorylates serine and threonine residues of its target proteins, activating them in the process. A-kinase is found in many different types of cells the target proteins in each kind of cell are different. Differences give rise to the variation of the responses to cAMP in different cells.

Example of cAMP as a second messenger: This diagram shows the mechanism for the formation of cyclic AMP (cAMP). cAMP serves as a second messenger to activate or inactivate proteins within the cell. Termination of the signal occurs when an enzyme called phosphodiesterase converts cAMP into AMP.

Present in small concentrations in the plasma membrane, inositol phospholipids are lipids that can also be converted into second messengers. Because these molecules are membrane components, they are located near membrane-bound receptors and can easily interact with them. Phosphatidylinositol (PI) is the main phospholipid that plays a role in cellular signaling. Enzymes known as kinases phosphorylate PI to form PI-phosphate (PIP) and PI-bisphosphate (PIP2).


How Does Insulin Signal a Cell to Take in Glucose?

A great (and well-used) example of a cell signaling pathway is seen in the balancing actions of insulin. Insulin, a small protein produced by the pancreas, is released when glucose levels in the blood get far too high.

First, the high glucose levels in the pancreas stimulate the release of insulin into the bloodstream. Insulin finds its way to the cells of the body, where it attaches to the insulin receptors. This sets off a signal transduction pathway within each cell that causes the glucose channels to open, as seen in this graphic:

As glucose flows into the cell, the glucose levels in the bloodstream are slowly decreased. The cells will use the glucose to create ATP energy or the cells store it as fats and starches for later use. Once the glucose level in the bloodstream has dropped to a sufficient level, the pancreas stops producing insulin, and the cells shut down their glucose channels.


Figure 3

Figure 3. The DIC images of CHO cells before, during, and after electroporation when a pulse of 800 V/cm and 25 ms was applied. (a) The image series that showed the dynamic change in the cell morphology throughout the electroporation process. (b) The images of cells before and 24 h after the application of the pulse. The cells that were live 24 h after electroporation are marked by red dots in the upper image.

In Figure 4, we examined the percent protein release from single cells when they were all exposed to a pulse of 800 V/cm and 25 ms (by comparing the fluorescence image of single cells before and after electroporation) and then compiled the histogram to link the protein release to the cell fate (live/dead). The cell population appears to roughly exhibit a normal distribution in terms of the percent release with the largest number of cells releasing a medium level of the protein (25–45%). However, there was no indication that a higher percent of protein release was associated with a higher probability for cell death at the single cell level. Counterintuitively, the cell death probability appears to be lower at the higher end of the protein release. This seems to suggest possible recovery mechanisms that are only triggered by larger amount of protein release and help maintain cell viability after the procedure. The details of such mechanisms will require further systematic studies.


In Situ Hybridization (ISH) and Fluorescence in Situ Hybridization (FISH)

In situ Hybridization (ISH) is a method that allows to localize and detect nucleic acid sequences within structurally intact cells or morphologically preserved tissues sections. Fluorescence in situ hybridization (FISH) is a kind of ISH which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity. The basic principles for FISH and all other methods of in situ hybridization are the same, except one is utilizing a fluorescence probe to detect specific nucleotide sequences within cells and tissues. They differ from immunohistochemistry which usually localize proteins in tissue sections. In situ hybridization can be performed on a variety of targets, including RNA within cells, DNA in metaphase chromosome preparations obtained from mitotic cells, or DNA in interphase nuclei from cells in the non-mitotic phases of the cell cycle. In addition to the advantages of detection in morphologic context, in situ hybridization has become popular also because of its high sensitivity in nucleic acid detection. In situ hybridization has been widely used for research applications, including clinical cytogenetics, gene mapping, tumor biology and studies of chromosome evolution.

Choice of Probe

Probe is critical to in situ hybridization, and a right probe can help you achieve your goals. Not only the probe types but also the label of probe should you take into account when you choose a probe for in situ hybridization.

There are essentially four types of probe that can be used in performing in situ hybridization. The information of the probe types is listed in Table1.

Table1 The information of probe types

Probe Types Advantages Disadvantages
Double-stranded DNA (dsDNA) probes Stable, available, easier to obtain Self-hybridize, less sensitive, need denaturation before hybridization
Single-stranded DNA (ssDNA) probes Stable, easier to work with, more specific, resistant to RNases, better tissue penetration, without self-hybridize Time consuming, expensive
RNA probes (riboprobes) Higher thermal stability, better tissue penetration, more specific, low background noise by RNase Sensitive to RNases
Synthetic oligonucleotides Economical, stable, available, easier to work with, more specific, resistant to RNases, better tissue penetration, better reproducibility Know the information of nucleotide sequence

To "see" where the probe has bound within your cells or tissue section you must attach a label to the probe before hybridization. The presence of the label should not interfere with the hybridization reaction. There are a variety of labeling techniques which can be divided into two types: radioactive isotopes and non-radioactive labels.

Radiolabeled probes, including 3 H, 35 S, 32 P, are still widely used due to its high activity which can be detected transcripts in low amounts. When using the radiolabeling, waste disposal and containment measures must be take into account and it must be noted that the useful shelf life of your labeled probe is inherently dependent on the half-life of the radionucleotide.

On the other hand, non-radioactive successfully used with in situ hybridization include digoxigenin (DIG), biotin and fluorescent labels. The non-radioactive labeled probe can be either used immediately or be stored at -20 °C as these non-radioactive labels have no inherent "decay" kinetics.

The process of the in situ hybridization can be divided into following steps:

Fig1. The process of genomic in situ hybridization (S P Brammer, et al, 2013)

1. Slide Preparation

For chromosome spreads, alcohol/ether (1:1) cleaned slides are sufficient. However, since tissue sections may be lost during the procedure, either polylysine or glutaraldehyde-activated gelatin chrome aluminum slides for these sections are required.

2. Sample Collection and Fixation

To preserve morphology, fresh tissue should be rapidly removed and fixed as soon as possible. From a chemical point of view, common precipitating fixatives (such as acetic acid/ethanol and Carnoy's fixative) are not recommended because of a fear that such fixatives would make the cell matrix impermeable, or alter the target nucleic acid to a point that hybridization would be reduced or prevented.

For metaphase chromosome spreads, methanol/acetic acid fixation is usually sufficient. For paraffin-embedded tissue sections, formalin fixation is always used. Cryostat sections fixed for 30 min with 4% formaldehyde or with Bouin’s fixative have been used successfully, as well as paraformaldehyde vapor fixation. There is still no fixation protocol which can be used for all substrates, and the fixation protocols must be optimized for different applications.

3. Embedding and Section

After fixation, the sample is embedded in paraffin or OCT for long-term storage and sectioning for subsequent procedure. The embedded tissues can be sectioned to thin slices with microtome or freezing microtome (15-20μm).

4. Permeablization

It is known that the target DNA or RNA sequences are surrounded by proteins and the extensive cross-linking of these proteins mask the target nucleic acid, which present obstacles to good infiltration of the probe. Therefore, permeabilization procedures are critical to in situ hybridization. Three main reagents used to permeabilize tissue are proteinase, HCl and detergents.

Protease treatment serves to increase target accessibility by digesting the proteins that surround the target nucleic acid. Proteinase K or pronase is usually used to remove those proteins. Optimal concentration has to be determined but a normal starting concentration is 1 μg/ml. Incubation has to be carefully monitored because if the digestion proceeds to far you could end up destroying most of the tissue or cell integrity.

In some protocols a 20-30 min treatment with 0.2M HCl is recommended. Although the precise action of the acid is unknown, the extraction of proteins and hydrolysis of the target sequence may contribute to a decrease of the level of background staining.

The Triton X-100, SDS and other detergents, are frequently used to permeabilize the membranes by extracting the lipids membrane. This is not usually required in tissue that has been embedded in wax, but it is critical to intact cells or cryostat sections.

5. Pretreatment/ Prehybridization

Pretreatment/ prehybridization is generally carried out to low the background noise.

When an enzyme (such as peroxidases or alkaline phosphatases) is used visualize the label, the endogenous enzymes which could result in high background have to be inactivated. This can be achieved with peroxidase by treating the sample with 1% H2O2 methanol for 30 min. As for alkaline phosphatase, the levamisole can be added to the substrate solution, however this may be unnecessary since the residual alkaline phosphatase activity is usually lost during hybridization.

RNase treatment serves to remove endogenous RNA and decrease background in hybridization reaction. It is performed by incubating the preparations in DNase-free RNase (100 μg/ml) in 2xSSC (SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.4) at 37°C for 60 min.

Prehybridization is carried out by incubating the tissue or section within a solution that contains all the components of the hybridization mixture, minus the probe. Not all protocols need this step.

6. Hybridization

Hybridization depends on the ability of the probe to anneal with a complementary target strand just below its melting point (Tm). Additionally, the composition of the hybridization solution is important to control the efficiency of hybridization process. The factors affecting the hybridization of the probe to the target sequence are:

  • monovalent cation concentration
  • temperature
  • organic solvents
  • pH
  • other components such as ssDNA, tRNA, ssDNA and polyA

Unbound or loosely bound probes are removed by performing washes. Solution parameters such as temperature, salt and detergent concentration can be manipulated to remove non-specific interactions.

8. Detection

As mentioned above, radiolabeled probes are detected by either photographic film or photographic emulsion. For non-radiolabeled probes, there are two methods: direct and indirect:


Watch the video: Intro to Cell Signaling (September 2022).


Comments:

  1. Malakus

    One and the same...

  2. Toby

    I think, that you are mistaken. Write to me in PM, we will talk.

  3. Nebar

    Not to tell it is more.

  4. Germano

    Now all is clear, thanks for an explanation.

  5. Moraunt

    You are making a mistake. I propose to discuss it.

  6. Bearach

    The message is removed

  7. Togquos

    Interestingly, I didn't even think about it ...



Write a message