Introduction to Cell Signalling and Techniques PDF

Cell Signalling in Health and Disease 6BH501 Dr Stella Ademowo [email protected] 26/09/2024 derby.ac.uk Body TitleOutline Session Text Arial Bold 32pt Example image can be Module introduction chang...

Cell Signalling in Health and Disease 6BH501 Dr Stella Ademowo [email protected] 26/09/2024 derby.ac.uk Body TitleOutline Session Text Arial Bold 32pt Example image can be Module introduction changed in the frame here Assessment requirements Concept of cell signalling Cell signalling techniques derby.ac.uk Body Title Text Arial Bold 32pt Introduction This module looks at Example image can be changed in the frame here communication between cells and its implication in health and disease derby.ac.uk Body Title Text Arial Bold 32pt Introduction Code – 6BH501 Example image can be changed in the frame here Time – Thursdays 9-12noon Delivery – On campus 20 credits (Autumn term) CW - 50%, Exam - 50 % BSc Biomedical Science, Biology, & Human Biology derby.ac.uk Body Title Text Arial Bold 32pt Resources Body Copy Arial 24pt Explore the course resources Announcement Module information Study materials Assessments Social Learning Reading list will be very useful for relevant materials Mid module feedback and module evaluation questionnaire (MEQ) derby.ac.uk Body Core Title Text Text Arial Bold 32pt Body Copy Arial 24pt derby.ac.uk Body Title Text Arial Bold 32pt Timetable derby.ac.uk Body Title Text Arial Bold 32pt Tutors Body Copy Arial 24pt Dr Stella Ademowo Dr Thomas Illingworth Dr Jinit Masania Dr Aparna Duggirala derby.ac.uk Body Title Text Arial Bold 32pt Assessment Body Copy Arial 24pt Formative: o Learning Science formative tutorials on signalling techniques (qPCR) o Quizzes Summative: Component 1 Full laboratory report on the practical session - analysis of gene expression (Weighting: 50%) Component 2 Written examination (Weighting: 50%) derby.ac.uk Body Title Text Assessment Arial and Bold 32pt deadlines Body Copy Arial 24pt Assessment Weighting Set date Submission Return Feedback date date Format Full laboratory report on gene 50% 30th Oct 4th Dec 8th Jan Feedback via expression Turnitin analysis L6 (1500 words) derby.ac.uk Body Title Text Reminder Arial Bold on Digital Skill 32pt Passport Body Copy Arial 24pt Document – Technical skills in the lab is in the programme page > Programme information tab Record need to be kept throughout the programme Module choices will affect the skills opportunities A pass/fail component of 6BY993 Research Project derby.ac.uk Body Title TextinArial Expectations Bold Digital 32pt Skill Passport Body Copy Arial 24pt Level 3 /4: Introduction to technical skills Evidence of competence at ‘basic level’ for 3 skills Level 5: Develop skills introduced at L ¾ and acquire new skills Evidence of additional competency for 3 further skills at ‘basic level’, and 3 skills at ‘intermediate level’ Industrial placement : demonstrate your progression through the placement module Level 6: Demonstrate ability to apply your skills (6BY993/6BY507) Evidence of additional competency for 2 further skills at ‘intermediate level’, and 2 skills at ‘advanced level’ derby.ac.uk Body WhatTitle Text is cell Arial Bold 32pt signalling? Body Copy Arial 24pt Cell signalling is the fundamental process by which specific information is transferred from the cell surface to the cytosol and ultimately to the nucleus, leading to changes in gene expression. Signalling cells release a molecule and targeted cell receives the signal derby.ac.uk Body Title Why do Textneed cells ArialtoBold 32pt communicate? Body Copy Arial 24pt To maintain stable internal environment (homeostasis) To regulate cell growth and division To organise cells into tissues To co-ordinate cellular functions Cellular mis-communications leads to onset and progression of diseases derby.ac.uk Body Title Text Importance Arialsignalling of cell Bold 32pt Body Copy Arial 24pt derby.ac.uk Body WhatTitle Textcell triggers Arial Bold 32pt signalling Body Copy Arial 24pt Change in activity or subcellular localization alters the ‘wiring’ of protein and initiates a signalling event Changes in cellular metabolism Changes in electrical charge across the plasma membrane Changes in the gene expression Components of cell signalling Ligands, receptors, second messengers, adapters, anchors, scaffolds, and transcription factors derby.ac.uk Body TitleofText Concept cellArial Bold 32pt signalling and signal transduction Body Copy Arial 24pt Receptor protein receives a signal, undergoes a conformational change, which in turn launches a series of biochemical reactions within the cell. Activation of receptors can trigger the synthesis of small molecules called second messengers, which initiate and coordinate intracellular signalling pathways. derby.ac.uk Body Title Modes Text Arial of cellular Bold 32pt communication Body Copy Arial 24pt Intercellular communication – Between cells Intracellular communication – Within a cell Plasma membrane-bound signalling molecules (receptors) for direct physical contact (A) Receptor protein inside target cells, signal protein must get into the cell (B) Receptors could form protein channels (gap junctions that co-ordinates activities of (C) GAP JUNCTION RECEPTORS adjacent cells) (C) derby.ac.uk Body Flow Title Text Arial Bold 32pt of information Body Copy Arial 24pt (C) GAP JUNCTION RECEPTORS derby.ac.uk Body Titleand Ligands Text Arial first Bold 32pt messengers Body Copy Arial 24pt Ligands are chemical groups which are able to bind to and effect receptor molecules; found intracellularly and extracellularly; activated in response to environmental demand What chemicals can act as cell signalling ligands? Hormones (Adrenaline) Cytokines (IL-6) Growth Factors (Endothelial growth factor) Neurotransmitters (Acetylcholine, serotonin) ATP (Adenosine triphosphate) derby.ac.uk Body Title Second Text Arial Bold 32pt messengers Body Copy Arial 24pt Second messengers bind to specific protein targets, altering their activity to relay downstream signals Direct binding of second messengers to targets, (usually enzymes), modify their catalytic activities Produced in response to extracellular and intracellular stimuli Their levels are controlled by various homeostatic mechanisms to ensure precision in cell signalling. derby.ac.uk Body Title Second Text Arial Bold 32pt messengers Body Copy Arial 24pt Intracellular release of 2nd messenger such as calcium, cAMP and a exchange of nucleotide can switch on signalling leading to diverse modifications (oxidation, phosphorylation, ubiquitinylation, methylation, acetylation, sulfation….) o Cyclic nucleotides and soluble molecules that signal within the cytosol o Lipid messengers that signal within cell membranes o Ions that signal within and between cellular compartments o Gases and free radicals that can signal throughout the cell and even to neighbouring cells. derby.ac.uk Body Title induced Hormone Text Arialcell Bold 32pt responses mediated by cAMP Body Copy Arial 24pt derby.ac.uk Body TypesTitle Text Arial Bold 32pt of receptors Body Copy Arial 24pt Cell surface receptors Ion channel - Ligand-gated or voltage-gated ion channels (Neurotransmitters, e.g acetylcholine, involved in muscle contraction, blood vessel dilation, body fluid secretion and heart rate regulation) G-protein linked receptor (Rhodopsin pigment in the retina) Enzyme coupled receptor (vascular endothelial growth factor receptor) Intracellular receptors (Inositol triphosphate receptor) derby.ac.uk Body TypesTitle Text Arial Bold 32pt of receptors Body Copy Arial 24pt derby.ac.uk Body Title Text Ion channel Arialreceptors linked Bold 32pt Body Copy Arial 24pt derby.ac.uk Body Title Text G-Protein Arialreceptor coupled Bold 32pt Body Copy Arial 24pt Along with the receptor protein, a G protein is associated with the function of the receptor G – proteins, or guanine nucleotide binding proteins, act as molecular switches. G – proteins contain 3 distinct domains, an α, β and γ subunits. Two of the three subunits are tethered onto the intracellular side of the plasma membrane through short lipid tails G – proteins are held in an inactive state until the GDP within the α subunit is substituted for a GTP group derby.ac.uk Body Titlecoupled Enzyme Text Arial Bold 32pt receptor Body Copy Arial 24pt Second group of transmembrane receptors that have intrinsic enzymatic activity. Act as an enzyme itself or it facilitates downstream signalling via enzyme linked activation of intermediate complexes. Enzyme coupled receptors o Receptor tyrosine kinases (RTK’s) o Receptor serine threonine kinases o Receptor tyrosine phosphatases o Receptor guanylyl cyclases RTK’s - largest group of ECRs, with the 58 known and 20 sub families derby.ac.uk Body Title tyrosine Receptor Text Arialkinase Bold 32pt Body Copy Arial 24pt RTK phosphorylation is regulated by protein tyrosine phosphatases (PTP), removing phosphates and deactivating the receptor following ligand release and returning it to the resting state derby.ac.uk Body Title tyrosine Receptor Text Arialkinase Bold 32pt Body Copy Arial 24pt RTK phosphorylation is regulated by protein tyrosine phosphatases (PTP), removing phosphates and deactivating the receptor following ligand release and returning it to the resting state derby.ac.uk  Body Title Texttyrosine Non receptor Arial Bold 32pt receptor kinase Body Copy Arial 24pt Different slightly from RTK’s in that ligand binding does not induce phosphorylation of the receptor in the first instance. Ligand binding leads to phosphorylation of the non-receptor kinase located on the cytosolic domain of the receptor. Once phosphorylated, the TK works as a protein kinase, phosphorylating the cytosolic domain of the receptor A non-receptor tyrosine kinase phosphorylates the receptor derby.ac.uk Body Title TextSignal Extracellular Arial Bold 32ptvia RTKs Proteins Body Copy Arial 24pt derby.ac.uk Body Title Textof Transduction Arial cell Bold 32pt signals Body Copy Arial 24pt Autocrine: Response to self signalling molecule (receptor on same cell) (Cancer cells, growth and clotting factors) Paracrine: Released by a cell and acts locally on another nearby cell (Cancer cells, growth and clotting factors) Endocrine: Secreted molecules pass through blood vessel to act on distant cells (hormones) Juxtacrine: Cell- cell; cell-extracellular matrix signalling that requires close contact usually adjacent cells (growth hormones, cytokines, chemokines) Syanptic: occurs between cells with a synapse (a specialized junction) that releases chemical neurotransmitters to post synaptic targets in neurons derby.ac.uk Body Title Textof Transduction Arial cell Bold 32pt signals Body Copy Arial 24pt derby.ac.uk Body Title Signal Text Arial Bold 32pt amplification Body Copy Arial 24pt derby.ac.uk Body Title Text Simplified Arial signal Bold 32pt pathway transduction Body Copy Arial 24pt derby.ac.uk Body Title Effects of Text Arial Bold 32pt cell signalling Body A Copy Arial 24pt strong local production of inflammatory mediators has systemic effects, reaching the whole body Post translational modifications The cellular signalling machinery makes homeostasis possible Mutated misfolded proteins causes cancer and some neurodegenerative diseases Synaptic dysfunction disrupts neuronal communication leading to AD and PD Type 2 diabetes develops in a series of stages from over nutrition derby.ac.uk  My Research – Healthy ageing Oxidative stress Inflammation Mitochondria function Cellular senescence Effect of antioxidants Schematic demonstrating the possible link between cellular senescence, altered lipid metabolism and organismal ageing Sensitivity: Internal Ademowo et al. 2017 Body Title Text Signalling Arial –Bold cascade 32ptsenescence cellular Body Copy Arial 24pt Permanent growth arrest derby.ac.uk Body Title Text Signalling Arial Bold 32pt cascade Autophagy Body Copy Arial 24pt derby.ac.uk  Interaction - PollsEverywhere PollEv.com/stellademowo Sensitivity: Internal Body Title Text Laboratory Arial Bold Detection 32pt Techniques in Cell Signalling Body Copy Arial 24pt Analysis of post translational modifications, protein-protein interactions and small molecule protein interactions Signals can be detected from any of the three levels of cell signalling Ligand Receptor Response derby.ac.uk Body Title Text Laboratory Arial Bold Detection 32pt Techniques in Cell Signalling Body Copy Arial 24pt Enzyme linked immunosorbent assay (ELISA) Western blotting Flow cytometry Protein arrays Immunohistochemistry Immunoprecipitation, Chromatin Immunoprecipitation ( ChIP) Omics technology / pathway analysis Immunofluorescence and confocal microscopy - Dr Illingworth Gene expressions (computational and experimental approaches) – Dr Aparna derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt Body Copy Arial 24pt Antigen: Any substance capable of eliciting an immune response Epitope: The small site on an antigen to which a complementary antibody may specifically bind, it is usually 1-6 monosaccharides or 5-8 amino acid residues on the surface of the antigen Antibody: An immunoglobulin capable of specific combination with the antigen that causes its production in a susceptible animal derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt Body Copy Arial 24pt Most antibodies are produced in mice, rats and rabbits, isolated and purified Primary antibodies detect specific proteins, e.g rheumatoid factor Monoclonal antibodies are produced from a single antibody producing B cell; this only binds with one unique epitope. Polyclonal antibodies are produced from different B cells; these recognise multiple epitopes on the same antigen Secondary antibodies are used to detect the primary antibody; these are raised against the species of the primary antibody e.g. anti mouse Some specific antibodies can recognise phosphorylation status derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt – Western blot Body Copy Arial 24pt Analysis of Immune activities– Western blotting permits the detection of proteins separated by gel electrophoresis 1D-gel electrophoresis Western blotting derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt – Western blot Body Copy Arial 24pt Phosphorylated p53 in human T lymphoblast derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt - ELISA Body Copy More Arial 24pt quantitative than western blot Micro plate-based assay utilising capture antibody specific for protein of interest (POI) POI either purified or in a complex heterogenous sample such as in cell lysate or biological fluid/extract is bound to antibody coated plate A detection antibody, specific for that interaction or phosphorylation site is added Fluorimetric or colorimetric readout Intensity of the resulting signal is directly proportional to the POI in the original sample. We can obtain quantifiable results by utilizing a calibrated standard High specificity due to the use of 2 antibodies specific for the POI in the sandwich format High sensitivity for small sample volumes and detection of low abundant proteins Microplate-based format allows for higher throughput than westernblot derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt - ELISA Body Copy Arial 24pt derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt – Flow cytometry Body Copy Arial 24pt Fluorescence-based assay that measures multiple characteristics, simultaneously (population counts, protein abundance, from individual cells suspended in a solution) Rapid, quantitative, and accurate measurement of cellular characteristics that provides unparalleled insight into the heterogeneity of cellular populations. Directs a single stream of cells past a light source and measures the resulting scatter and emission of light energy in various wavelengths Fluorescent molecules with different excitation and emission characteristics are combined, detected, and differentiated to provide multiple readouts from a single cell Fluorescent molecules may be attached to antibodies targeting specific proteins or protein modifications, or they may be dyes that directly bind to cellular components derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt – Flow cytometry Body Copy subpopulation Complex Arial 24pt phenotyping and cell signalling analysis in cell suspension or biological fluids Quantitative analysis at a single cell level, to gain understanding at the cellular level and/or at the population level. Activity of 2 key signalling pathways in 3 immune cell types changes in response to a treatment could be detected within a few hours by labelling and running a single sample on a flow cytometer Antibody or fluorophore-based detection of intracellular cell signalling derby.ac.uk Body Title Text Arial Antibody-based Bolddetection protein 32pt – Protein array Body Copy Arial Analyse 24pt multiple proteins and their modifications, protein-protein interactions at the same time A global view of a biological system for better understanding of entire networks or pathways. We now have protein arrays, which can analyse up to 20,000 proteins. derby.ac.uk Body OtherTitle Text Arial detection Bold 32pt techniques Body Copy Arial 24pt Spatial distribution of cells in tissue can be investigated using immunohistochemistry or tissue-based immunofluorescence Fine-scale subcellular analysis is more suited to analysis by fluorescent and/or confocal microscopy Analysis of genetic mutations or large-scale mRNA analysis is better left to sequencing instruments Omics technology (Proteomics, genomics, lipidomics, metabolomics, glycomics) derby.ac.uk  Summary Cells can signal to each other via ligands, which bind to cellular membrane receptors or internal receptors within the cell. Signals may be transduced through the cell via a series of secondary messenger molecules and proteins. Many cell signalling pathways result in a change in gene expression, where their activation directly influences the activity of regulatory transcription factors. We can use numerous laboratory/computer-based techniques to explore cell signalling pathways. Cell signalling is important for the maintenance of cellular homeostasis, dysregulation of these pathways can result in diseases. Sensitivity: Internal https://www.youtube.com/watch?v=-dbRterutHY  Questions Sensitivity: Internal