Cell and Molecular Biology Lecture Finals 2024-2025 PDF

CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CELL SIGNALING YEAST CELLS COMMUNICATE TO MATE IRRITABILITY Saccharomyces cerevisiae multiply by Seismonastic movements...

CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CELL SIGNALING YEAST CELLS COMMUNICATE TO MATE IRRITABILITY Saccharomyces cerevisiae multiply by Seismonastic movements budding not until a cell is ready for mating ○ Pulvini cells gain and lose turgor due thus sending “mating factors” to the other to water movement in and out of cell to stop budding and instead perform these cells. conjugation ○ Opening of ion channels resulting in Diploid zygote is an opportunity to combine movement of K, Cl, and Ca ions lead genetic material and shuffle during spore to rapid decrease in water in the formation (2n to n) pulvini. CELLS EXHIBIT IRRITABILITY Respond to external and internal stimuli Response to stimuli affects their structure, growth, reproduction, development, differentiation, and behavior. Protein receptors receive signals A means to communicate with other cells COMMUNICATION IN MULTICELLULAR ORGANISMS Use chemical signals Signal is received by a receptor (either a membrane or cytosolic protein) Activation of intracellular signaling pathways or systems Effectors: transcription factors, ion channels, metabolic enzymes, signaling proteins, cytoskeletons. QUORUM SENSING Coordinated behavior in bacteria at higher population density as a result of chemical signals. May result to coordinated motility, antibiotic production, spore formation or sexual conjugation INTERCELLULAR SIGNALING RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 AUTOCRINE STIMULATION CELLS RESPOND TO MULTIPLE EXTRA-CELLULAR SIGNALS Extracellular signals are either soluble molecules, bound to extracellular matrix, or found in the surface of other cells Specific combination of signals will lead to specific cellular response Cells respond selectively to signals essential for regulation of cellular activities. Cells undergo programmed cell death in the absence of signals SIGNAL RECEPTORS Extracellular signals are diverse (proteins, EXTRACELLULAR SIGNALS INDUCE peptides, amino acids, nucleotides, CELL-SPECIFIC RESPONSE steroids, retinoids, dissolved gasses) An extra-cellular signal (e.g. Acetylcholine) Signals are released from signaling cells by may bind to receptors on different cells to exocytosis or diffusion induce different cellular responses. Cell surface receptors mostly recognized Cellular response depends on activated big hydrophilic signals signaling proteins, effector proteins, and Intracellular receptors mostly recognize genes that are activated in a particular cell hydrophobic small molecules (predetermined developmental history and Signals are often at very low gene expression profile) concentrations but with high affinity binding to receptors. CLASSES OF CELL-SURFACE RECEPTORS Ion-channel-coupled receptors G-protein-coupled receptors Enzyme-coupled receptors ○ Membrane bound proteins with an extracellular domain for signal (ligand) binding and signals do not enter the cytosol. ○ Act as signal transducers – convert the ligand-receptor binding event into intracellular signals which will produce a cellular response. ○ Highly specific interaction with a ligand (extracellular signal) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ION-CHANNEL-COUPLED RECEPTORS 30-50% of human proteins contain Transmitter-gated ion channels or phosphates ionotropic receptors (often are multi pass Human genome encodes about 520 protein channels) kinases and 150 protein phosphatases Involved in rapid synaptic signaling Serine/Threonine Kinases phosphorylate between a pre- and post-synaptic cells the hydroxyl groups of serines and (nerves, muscles). threonines Signals are often neurotransmitters that Tyrosine kinases phosphorylate tyrosines induces an “open” or “closed” state of an Kinase Cascades – series of kinases that are ion channel (changes the excitability of the activated one after the other. post-synaptic cell) G-PROTEIN-COUPLED-RECEPTORS Membrane-receptor interacts with a trimeric GTP-binding protein (G-Protein) after the binding of a signal molecule Activated G-protein will induce the activation of a target protein (either an enzyme or a channel) Leads to change in the concentration of intracellular signaling molecules (if target GTP-binding proteins as second protein is an enzyme) or change ion messengers permeability of plasma membrane (if target Turned ON when GTP bound protein is a channel) Turned OFF when GDP bound GTP hydrolysis involves removal of a phosphate molecule from GTP to become GDP 2 types: ○ Trimeric GTP-binding proteins ○ Monomeric GTP-binding proteins ENZYME-COUPLED RECEPTORS The receptor is either a multi-domain protein with catalytic domain or it may interact with an associated enzyme. Single pass membrane proteins with ligand-binding domain on the extracellular side and a catalytic domain or enzyme -binding site on the cytosolic side. Mostly protein kinases or proteins that associate with protein kinases (involved in phosphorylation of specific proteins) SECOND MESSENGERS AS MOLECULAR REGULATION OF GTPASE SWITCHES GTPase-activating proteins (GAPs) Phosphorylation is a process of activating hydrolyses the GTP thus turning OFF the or deactivating second messengers GTPase Protein kinases (add phosphate) and Guanine nucleotide exchange factors protein phosphatases (removes phosphate) (GEFs) activate GTP-binding proteins by turn ON and OFF second messengers. releasing GDP and adding GTP thus turning ON the GTPase RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Receptor is auto phosphorylated after signal binding Phosphorylated sites serve as docking sites for intracellular signaling proteins. Rapid disassembly after signaling INTRACELLULAR SIGNALING COMPLEXES (3) Involves the phosphorylation of phospholipids (Phosphoinositides) SIGNALING PATHWAY IS A SERIES OF attached to the plasma membrane once ACTIVATION signal molecule binds to the receptor Simple signaling involves the Inactive intracellular signaling proteins phosphorylation of an inhibitor protein to bind to phosphorylated phospholipids to release and activate a transcription activate downstream signaling regulator to allow gene expression Two sequential inhibitory signals produces a positive signal for gene expression (double-negative activation) SIGNALING COMPLEXES ARE FORMED USING MODULAR INTERACTION DOMAINS INTRACELLULAR SIGNALING COMPLEXES (1) Induced Proximity – activation of second Scaffold Proteins – groups of interacting messengers by bringing them close together signaling proteins to ensure efficiency, They use their small interaction domains to specificity, and robustness of response and be activated prevent crosstalk between signals ○ Src homology 2 (SH2) domain – Pre-assembled complex of proteins bind to Proline-rich aa sequences interacting with the receptor ○ Phosphotyrosine-binding (PTB) domain – bind to phosphorylated Tyr ○ Pleckstrin homology (PH) domain – bind to charged head groups PI Example: Insulin receptor INTRACELLULAR SIGNALING COMPLEXES (2) Transient assembly of signaling proteins after signal binding to receptor. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 FEATURES OF SIGNALING SYSTEMS Response timing – length of time before a cell produces a response after the binding of a signal to its receptor (ranges from milliseconds to days) Sensitivity – a measure of cellular response based on amount of signal needed (from low to high concentration of signals). Self-amplification affects the sensitivity of signaling systems. Dynamic range – the range of signals that a system requires to illicit a cellular response (narrow or broad range of signals). May be affected by adaptation mechanism. Persistence – a measure of the duration of the interaction between a signal (ligand) to IMPORTANCE OF RAPID TURNOVER OF SIGNAL its receptor (ranges from transient MOLECULES interaction to prolonged or repeated The concentration of signal molecules that interactions including positive feedback). are degraded rapidly change very quickly Signal processing – complexity of the (red lines) regardless of the decrease or interaction of second messengers after increase in the rate of synthesis. signal binding (sometimes oscillatory The concentration of signal molecules that response – series of responses) will degrade slowly (green lines) change Integration – ability of a cellular response proportionally more slowly. to be governed by multiple inputs (several signals binding to different receptors but the effect is the same thus the need for coordination) Coordination – ability to produce multiple response from a single signal thru the activation of multiple effectors creating branching pathways of signaling. SIGNAL INTEGRATION Some cellular responses are the results of integrated signaling pathways. 2 or more different signals binding to different receptors and converge to have a common cellular response Coincidence detectors – proteins involved in the integration of a response Examples: ○ Cell survival and proliferation DIFFERENCES IN CELLULAR RESPONSE Some cells respond by generating smoothly graded response (hyperbolic response) over a wide range of extracellular signal concentrations Some cells respond slowly at low concentrations of signals and produces steeper response at intermediate signal concentrations (sigmoidal response). Some cells respond abruptly to minimal signal concentrations (cell response from low to high responses called all-or-none response) TURNOVER OF SIGNAL MOLECULES AFFECTS THE SPEED OF RESPONSE Turnover of the signal molecule refers to the destruction of the signal molecule or its speed of recycling. Also affected by the rate of synthesis of the signal molecule. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 to “on’ and “off” even in the removal of the EFFECT OF NUMBER OF SECOND MESSENGERS IN signal CELLULAR RESPONSE Summary of cellular response when the signaling involves the binding of 1 (green), 2 (blue), 8 (red), and 16 (black) effector molecules. Sharpness of activation response is a function of allosteric effector molecules that must bind to a target protein. If number of effector molecule is large enough, an all-or-none response is generated (black) EFFECT OF NEGATIVE FEEDBACK A Negative Feedback activates the I phosphatase thus deactivating the E kinase. Cellular response is observed only while the signal is bound the the receptor Presence of negative feedback but with a delay produces a brief response then goes back to normal level Presence of long delay in the negative FEEDBACK MECHANISMS IN CELL SIGNALING feedback produces sustained oscillation in Positive Feedback produces end results that cellular response. will activate further the signaling ○ Moderate strength feedback generates sigmoidal response ○ Strong feedback generates all-or-none response Negative Feedback produces end result that inhibits the signaling process EFFECTS OF POSITIVE FEEDBACK SIGMOIDAL RESPONSE MAY BE EXPLAINED IN 2 Positive Feedback produces a self WAYS sustaining response which may persist even Experimental result showing sigmoidal after the signal strength drops. response in MAP Kinase activation when Condition is called bistable frog oocytes were treated with increasing Without a positive feedback, the activity of progesterone levels E kinase is proportional to the level of Possibility 1: every oocyte in the population stimulation (binding of signal to receptor) gradually increased their MAP Kinase With positive feedback the transient activity thus the sigmoidal response stimulation of signal kinase turns he system RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Possibility 2: the oocytes registered G-PROTEINS all-or-none response one after the other Trimeric GTP-binding proteins associated with a GPCR 3 subunits (⍺ subunit, β subunit, and 𝛾 subunit) Inactive form when ⍺ subunit is bound to GDP CELLS CAN ADJUST THEIR SENSITIVITY TO A SIGNAL Different ways on how target cells can become adapted (desensitized) to extracellular molecules. ACTIVATION OF G-PROTEINS Binding of ligand to GPCR allows the receptor to act like a guanine exchange factor which will induce conformational change on the ⍺ subunit of G-protein to release GDP and add GTP GTP-bound G-protein is in active state ready to activate effector molecules (either an enzyme or an ion channel). G-PROTEIN COUPLED RECEPTORS Activated ⍺ subunit releases and activates Most common and most diverse receptor β-𝛾 subunit complex which could activate in terms of signals (ligands) recognized. other effector molecules Single polypeptide chain with 7 trans-membrane domains. Deep ligand-binding site at the center of the extracellular side Interacts with a G-protein on the cytosolic side More than 800 GPCRs in humans and about 1000 GPCRs are related to sense of smell alone in mice. Ligands – hormones, neurotransmitters (adrenalin activates 9, acetylcholine activates 5, serotonin activates ~14 distinct GPCRs), peptides, derivatives of fatty acids and amino acids, light, and molecules with smell and taste. Half of all known drugs bind to GPCRs. 150 orphan GPCRs (no known ligands). CYCLIC-AMP AS SECOND MESSENGER cAMP an example of second messenger in cell cell signaling cAMP is catalyzed by adenyl cyclase from ATP as a substrate though the removal of pyrophosphate cAMP is unstable and immediately hydrolyzed by cAMP phosphodiesterase to produce 5’ AMP RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 GS-STIMULATED INCREASE IN cAMP cAMP-DEPENDENT PROTEIN KINASE cAMP activates cAMP-dependent Protein Kinases (PKAs) cAMP CAN BE RAPIDLY INCREASED PKAs phosphorylates Serines or Threonines on target proteins ~20-fold increase per second after Regulatory subunit (A kinase) is bound to activation catalytic subunit in an inactive form. Neurons can produce 10-6 M after ligand Binding of cAMP to regulatory subunit binding (red and yellow signals indicate releases and activates catalytic subunit. high and intermediate levels of cAMP). Blue A-Kinase Anchoring Protein (AKAP) – fluorescence indicate low level of cAMP binds the regulatory subunit to the expression cytoskeletons. TYPES OF G-PROTEINS Stimulatory G-Proteins (Gs) – activates adenylyl cyclase for cAMP production cAMP INCREASE MAY RESULT TO GENE Inhibitory G-Proteins (Gi) – inhibits TRANSCRIPTION adenylyl cyclase thus inhibiting cAMP Increase in cAMP levels in the cytosol will production result to activation of PKA Cholera toxin – transfers ADP ribose from PKA activates CRE-binding (CREB) proteins NAD+ to Gs (ADP ribosylation) which alters Activated CREB recruits CBP before binding the ⍺ subunit that it cannot hydrolyse GTP to cyclic AMP response element (CRE) of (remains active) which stimulates adenylyl somatostatin gene promoter region cyclase to keep on producing cAMP. Results Results to transcription of somatostatin to efflux of Cl, Na and H2O causing gene. diarrhea. Translation of somatostatin can transform Pertussis toxin – catalyses the ADP short cAMP signal to long-term change in ribosylation of ⍺ subunit Gi preventing the cell which is important part of learning and interaction of G-protein with GPCR thus Gi memory. remains in inactive form RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 THE ROLE OF CALCIUM IONS Intracellular mediator of signal transduction Triggers muscle contraction Induces exocytosis of neurotransmitters in nerve cells Causes secretion of products in secretory cells Opens certain membrane channels (e.g. ryanodine receptor) G-PROTEINS SIGNAL VIA PHOSPHOLIPIDS Many GPCRs activate G-Proteins which in turn activates membrane bound enzyme Phospholipase C-β (PLCβ). ACTIVATION OF PLCβ Phospholipase C-β (PLCβ) – breaks PIP2 into diacyglycerol (DAG) and Inositol Triphosphate (IP3) DAG and IP3 are second messengers activating different signaling pathways. CALCIUM WAVES IN FERTILIZED EGG Fusion of sperm cell to an egg cell causes a wave of Ca2+ from the point of entry of sperm across the entire fertilized egg Changes the egg surface to prevent entry of other sperm cells. Ca2+-induced Calcium Release (CICR) – result of positive feedback Initial Ca2+ release starts from the sperm cell using Phospholipase C-zeta (PLC-ζ) which cleaves PIP2 to produce IP3 to release Ca2+ from ER. ACTIVITY OF IP3 AND DAG IP3 binds to its receptors (IP3-gated ion channels) on the surface of ER to release Ca2+ ions DAG and Ca2+ activates Protein Kinase C (PKC), a Ca2+-dependent kinase : RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CALCIUM WAVES AND OSCILLATIONS CALMODULIN:A CA2+- BINDING PROTEIN Positive and negative feedback produces Ubiquitous protein in eukaryotic cells that Ca2+ waves and oscillations. may constitute up to 1% of a cell’s total Presence of small amount of Ca2+ initiates protein mass. opening of IP3 channels (Ca2+ channels) Functions as intracellular Ca2+ receptor It will generate a wave of Ca2+ release by With 4 Ca2+ binding sites serially activating inactive IP3 channels Active state when bound to Ca2+ (displays (red) to open (green) sigmoidal response with increasing High concentrations of Ca2+ generates concentrations of Ca2+). negative feedback to inactivate the IP3 It binds to other effector proteins (enzymes channels thus inhibiting Ca2+ release and transport proteins) once activated. CA2+- CALMODULIN-DEPENDENT KINASES CaM Kinase II – a 12 enzyme complex arranged into 2 ring stacks (6 enzymes on each ring) Each monomer contains kinase domain, hub domain and regulatory segment (with linker and phosphorylation site) Binding of Ca2+-Calmodulin activates one kinase enzyme Autophosphorylation of nearby kinase VASOPRESSIN-INDUCED CA2+ OSCILLATIONS monomer. Liver cells were loaded with Ca2+ sensitive Prolonged enzyme activity is cause by aquorin and were exposed to increasing trapping of Ca2+-Calmodulin complex and concentrations of vasopressin. converts to enzyme to Ca2+ independent Activation of GPCR which activates PLCβ form. Spikes of Ca2+ release are observed with every treatment of vasopressin Frequency, amplitude and breath of oscillation reflects the signal strength. Other factors that may affect oscillations include phosphorylation, (affects Ca2+ sensitivity), other properties of signaling molecules. CaM-KINASE II ACTIVITY IN CA2+ OSCILLATIONS CaM Kinase II as a frequency decoder of Ca2+ Oscillations At low frequency Ca2+ spikes, CaM-kinase activity immediately goes down after every spike (goes back to inactive state). At high frequency Ca2+ spikes, autophosphorylation of enzymes results to continuous CaM-Kinase II activity (enzymes fail to inactivate completely) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 cyclase and degraded by cyclic GMP phosphodiesterase). Inner and outer segments of rod photoreceptor are specialized parts of primary cilium – serves as a signaling organelle. G-PROTEINS ALSO REGULATE OTHER PROTEINS Special type of G-Protein called G12 activates GEF which activates Rho family of protein which in turn regulates actin cytoskeletons. Other G-proteins regulate ion channels thereby altering ion permeability and electrical excitability. ○ Example: Opening of K+ muscarinic acetylcholine receptors in heart muscles) Activation of cyclic-nucleotide-gated ion channels involved in sense of smell and sight. OLFACTORY RECEPTORS ARE GPCRS Odorant binding to a GPCR activates G Protein (Golf) 350 GPCRs in humans, ~1000 GPCRs in mice Animals detect pheromones via different RESPONSE OF ROD PHOTORECEPTOR TO LIGHT GPCRs Light activates rhodopsins via conformational change of opsin Activation of Gt (G-Protein) Activation of cGMP phosphodiesterase Decrease in the levels of cGMP Closing of cGMP cation channels (light signal is converted to electrical signal) Hyperpolarization of rod cell plasma membrane MECHANISMS OF SENSE OF SMELL Odorant binding to a GPCR Activation of Golf Activation of Adenylyl cyclase 4. Increase in levels of cAMP Opening of cAMP gated cation channels Influx of Ca2+ Depolarization of olfactory receptor neuron Sending of nerve impulse to the brain VERTEBRATE VISION INVOLVES GPCRS Rod photoreceptors (non-color vision in dim light) and Cone photoreceptors (color vision in bright light) – are stimulated by light. Rhodopsin is a GPCR stimulated by photon. Photoreception is the fastest known G-protein mediated response in vertebrates. CONTROL OF RHODOPSIN ACTIVATION Activates G-Protein transducin (Gt). Rhodopsin Kinase – phosphorylates Photoreception transduction of signal uses rhodopsin to make it inactive – part of cyclic GMP (synthesized by guanylyl negative feedback loop to control protoreception. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Arrestin – inhibitory protein that binds to Viagra is a PDE5 inhibitor thus blocking the phosphorylated Rhodopsins degradation of cGMP resulting to prolonged penile erection. 4 MAJOR FAMILIES OF TRIMERIC G PROTEINS NITRIC OXIDE AS A SIGNALING MEDIATOR APPLICATION OF SIGNALS Nitric Oxide (NO) is a hydrophobic and a Signal transduction involves relay chains of small molecule that it can easily pass proteins and second messengers. through the bilipid membrane. Every part of the relay chain is an Acts as a signal molecule in animal and opportunity to amplify the signal. plant cells. Applicable to all GPCR binding signals Functions of NO A single signal is enough to activate a ○ Relaxes smooth muscle contraction cellular response. in blood vessels resulting to blood Cells are equipped with control vessel dilation. mechanisms along every step to regulate ○ Stimulates synthesis of cGMP by the process. binding reversibly to iron in the active site of guanylyl cyclase ○ Alters the activity of certain intracellular proteins by covalently nitrosylating thiol (-SH) groups on specific cysteines. NO is produced from the deamination of Arginine catalyzed by NO Synthase (NOS) ○ eNOS – NO synthase in endothelial cells ○ nNOS – NO synthase in neurons and neuro-muscular junctions ○ iNOS – NO synthase in macrophages (immune cells) NITRIC OXIDE IN SMOOTH MUSCLE RELAXATION GPCR DESENSITILIZATION Phosphorylation of GPCR by GPCR Kinase (GRK) makes the receptor desensitized (adapted) through the binding of arrestins. MECHANISM OF ACTION OF VIAGRA Important aspect of cellular regulation. NO activates guanylyl cyclase to produce 3 Modes of Desensitization of GPCRs: cGMP which relaxes smooth muscles in ○ Receptor Sequestration – penile blood vessels. temporary endocytosis (no access to Phosphodiesterase 5 (PDE5) degrades ligand) cGMP (converts to GMP) to reverse the process of smooth muscle relaxation. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ○ Receptor Down-regulation – ACTIVATION OF RTKS BY DIMERIZATION internalization followed by Extracellular signal proteins bind to lysosomal degradation dimerized RTKs. ○ Receptor Inactivation – structural Trans-autophosphorylation activates the change to prevent inaction with G dimerized RTKs proteins Activated RTKs creates binding sites (docking sites) for signaling proteins Bound signaling proteins transduce the signals to effector or second messengers ENZYME COUPLED RECEPTORS Integral/transmembrane (often single transmembrane domain) proteins with ligand binding domain on the extracellular SOME RTKS FOR ASYMMETRIC DIMER side and a catalytic domain or EGF Receptor Kinase for asymmetric enzyme-binding domain on the cytosolic dimers side Exist as monomers in inactive form May activate the same signaling pathways (absence of EGF) with GPCRs. Presence of Activator and Receiver enzymes in asymmetric dimers Activator enzyme phosphorylates the tyrosine residues of both enzymes RECEPTOR TYROSINE KINASES (RTKS) PROTEINS WITH SH2 DOMAINS BIND TO ACTIVATED RTKS Binding of SH2 (SRC Homology Region) containing proteins in platelet-derived growth factor (PDGF) receptor. Kinase Insert Region contains 3 binding sites while C-terminus tail has 2 binding sites Specific proteins bind to specific binding sites (e.g. PLC𝛾 binds to phosphorylated 1009 and 1021 tyrosines (determined by mutagenesis) SUBFAMILIES OF RTKS Groupings based on structures of extracellular domain. Common features of cytosolic domain – with tyrosine kinase domain Binding of ligand leads to phosphorylation of tyrosine kinase domain (activated form) ~60 human RTKs (divided into 20 structural subfamilies) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 GTPASE RAS MEDIATES SIGNALING BY MOST THE MAP KINASE MODULE RTKS Ras-GTP activates the Mitogen Activated Ras and Rho superfamilies of monomeric Protein Kinase Module (MAP Kinase GTPases relay signals from cell surface Module) with 3 components: receptors. ○ MAP Kinase Kinase Kinase ○ Ras (rat sarcoma virus) (MAPKKK) = Raf (Rapidly activated ○ Rho (Ras homologous) fibrosarcoma) ○ ARF (ADP Ribosyl Factor) ○ MAP Kinase Kinase (MAPKK) = Mek ○ Rab (Ras associated binding protein) (MAPK/Erk Kinase) ○ Ran (Ras related nuclear protein) ○ MAP Kinase (MAPK) = Erk (extracellular signal regulated kinase) ACTIVATION OF RAS PROTEINS Ras GTPase-activating proteins (Ras GAPs) hydrolyses GTP bound to Ras to inactivate the protein. MAP KINASE MODULE IN SCAFFOLDS IN YEASTS Different kinases are involved in MAP Kinase Modules to elicit different responses. Grouped into scaffolds to prevent crosstalk Activation of MAP Kinase Module may be triggered by different ligands binding to Binding of signal activates RTK different receptors (autophosphyration of Tyrosine amino acids) Grb2 adaptor protein binds to phosphorylated tyrosine on RTK via SHS domain SH3-bound Ras-GEF (Sos, son-of-sevenless) activates inactive Ras via removal of GDP and binding of GTP Activation of Ras protein (GTP-bound) TRANSIENT ACTIVATION OF RAS Ras linked to Yellow Fluorescence Protein (YFP) was expressed in human cancer cell lines. Cell line was injected with GTP labelled RHO FAMILY OF GTPASES with Red Fluorescent dye and activated Rho (Ras Homology) proteins are with EGF. monomeric GTPases that regulate actin and GTP binds to Ras transferring the yellow microtubules to control: fluorescence to the red dye (Fluorescence ○ Cell shape Resonance Energy Transfer, FRET) which ○ Cell polarity peaks at 3 mins and immediately goes down ○ Motility and migration indicating transient activation. ○ Adhesion ○ Cell-cycle progression ○ Gene transcription ○ Membrane transport Ligand Ephrin A1 binds to RTK (EphA4) Tyrosine Kinase activates Ephexin (Rho GEF) Ephexin activates RhoA (removes GDP and add GTP) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Myosin-mediated actin filament ACTIVATION OF mTOR contraction (growth cone collapse) Mammalian Target of Rapamycin (mTOR) is activated PI 3 Kinase – Akt signaling pathway to promote cell growth. Absence of extracellular growth factors will keep the Rheb inactive (GDP bound) via active Tsc2 inhibition. Binding of growth factors will activate the PI 3 Kinase –Akt pathway leading to inactivation of Tsc2 Activation of Rheb (GTP-bound) Activate mTOR PI 3 KINASE PRODUCES LIPID DOCKING Phosphoinositide 2 Kinase phosphorylates: OVERLAPS IN SIGNALING PATHWAYS ○ PI to PI(3)P ○ PI(4)P to PI(3,4)P2 Parallel signaling pathways occur inside ○ PI(4,5)P2 to PI(3,4,5)P3 the cell to illicit a common response. PI(3,4,5)P3 will serve as docking sites for Underscores complexity of signal intracellular signaling proteins. transduction Complex cellular responses involves several signaling pathways PI 3 KINASE SIGNALING PROMOTES CELL SURVIVAL Insulin-like Growth Factors (IGF) promotes CYTOPLASMIC TYROSINE KINASES cell growth via PI 3 Kinase pathway Src (sarcoma)family of cytoplasmic tyrosine Activated PI 3 Kinase produces PI(3,4,5)P3 kinases – largest group of tyrosine kinases which serves as docking sites for PDK1 in mammalian cells. (phosphoinositide-dependent Protein Contains SH1 to SH4 domains. Kinase 1) and Akt (a serine/threonine Contain highly conserved sequences. kinase) Phosphorylation of Bad (BCL2 associated agonist of cell death) activates the apoptosis inhibitory protein RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CYTOKINE RECEPTORS ACTIVATES JAK-STAT SIGNALING Cytokines are secreted small proteins that control the growth and activity of immune cells (interleukins, chemokines, interferons, lymphokines, etc). Cytokine Receptors (mostly dimers) stably associate with Janus Kinases (Jaks) which phosphorylate and activate STATs (Signal Transducers and Activators of Transcription) 2 Jaks cross-phosphorylate each other followed by phosphorylation the of cytokine receptor (to serve as binding site of STAT) and STAT Dimerized STAT is transported to nucleus to act as a transcription factor. REGULATION OF SMAD PATHWAY Negative feedback for the production of inhibitory Smads (Smad6 and Smad7) Mechanisms: ○ Inhibitory Smads – competes with R-Smads (Smad2 and Smad 3) to bind to receptors (decreasing phosphorylation. ○ Recruitment of ubiquiltin ligase called Smurf (Smad Ubiquitylation regulatory factors) to add ubiquitin to TGFβ receptors leading to EXAMPLES OF JAK-STAT SIGNALING internalization of receptors for degradation. Negative feedback controls JAK-STAT ○ Recruitment of phosphatases to Signaling via activation by STAT of dephosphorylate receptors inhibitory protein transcription. Also achieved by dephosphorylation thru protein NOTCH RECEPTOR AND DELTA LIGAND phosphatases. Neural cell development from epithelial cells involves sending of Delta signal from the differentiating neural cell to the Notch receptors in neighboring epithelial cells Lateral inhibition - the sending of signal is a means of communication to tell neighboring cells not to do the same differentiation. SMAD-DEPENDENT SIGNALING PATHWAY BY TGFβ Transforming Growth Factor β (TGFβ) – dimeric proteins acts as hormones or mediators of signaling involved in cell proliferation, differentiation, ECM production and cell death. TGFβ Receptors are tetrameric enzyme coupled receptors with serine/threonine kinase domain (e each of type 1 and type II receptors). Type II receptors phosphorylates type I receptors which will phosphorylate Smad 2 or Smad3 Formation of trimeric Smad complex which will enter the nucleus to be a part of PROTEOLYTIC CLEAVAGE OF NOTCH RECEPTOR transcription regulatory complex for the Notch receptor is a single pass transcription of a target gene. transmembrane protein with 3 proteolytic cleavage sites Needs to undergo proteolytic cleavage to function. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 First cleavage occurs in TGN membrane as a HEDGEHOG SIGNALING part of post-translational modification. Hedgehog proteins – when mutated Binding of Delta ligand (via EGF like produces Drosophila larvae with spikes domains) triggers cleavage 2 and 3 with the (denticles). action of 𝛾-secretase. Hedgehog signaling is associated with the Notch tail migrates to nucleus and will bind development of basal cell carcinoma of the to Rbpsuh protein converting it from skin in humans. transcriptional repressor to transcriptional activator SMALL HYDROPHOBIC SIGNALS BIND TO INTRACELLULAR RECEPTORS Diffuse directly to plasma membrane and WNT LIGAND AND FRIZZLED RECEPTOR bind to intracellular receptors (often Wnt protein is a secreted morphogen transcription regulators) involved in several aspects development such wings (in Drosophila) and breast tumors (in mice) Frizzled Receptor – 7multi-pass transmembrane receptor binds to Dishevelled protein In the absence of Wnt, β-catenin is degraded by proteasomes as a result of phosphorylation by Casein Kinase 1 (CK1) and a second phosphorylation and ubiquitylation by Glycogen Synthase ACTIVATION OF NUCLEAR RECEPTORS Kinase 3 (GSK3) Nuclear Receptors are inactive when bound Axin and APC (adenomatous polyposis coli) to inhibitory protein – form the scaffold protein 3 domains: LRP (LDL-receptor related protein) ○ Transcription-activating domain interacts with Frizzled receptor upon ○ DNA-binding domain binding of Wnt ○ Ligand binding domain Dephosphorylated β-catenin moves to Needs coactivator protein to start nucleus to remove Groucho transcription (co-repressor)and binds to LEF1/TCF to initiate transcription of target genes. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CICARDIAN CLOCK MAY CONTROL GENE EXPRESSION Circadian Clock – refers to the natural internal rhythms of organisms in response cycles of day and night. Circadian clock which contains a negative feedback loops that control gene expression Accumulation and decay of transcription regulatory proteins - TIM (timeless) and PER (Period) mRNAs of TIM and PER rise gradually during the day. PER inhibits transcription of TIM and PER AUXIN SIGNALING PATHWAY Auxin is indole-3 acetic acid Auxin binds to a auxin receptor protein and a transcriptional repressor (AUX/IAA) Auxin response factor (ARF) is inactive in the absence of auxin as a result of binding od AUX/IAA AUX/IAA-Auxin Receptor complex is ubiquitynalated and degraded in the PLANTS AND ANIMALS USE DIFFERENT CELL presence of Auxin SIGNALING Divergence of plants and animals over more than a billion years ago indicates differences in mechanisms of cell signaling (no homologs for many animal signaling proteins in plants). RECEPTOR SERINE/THREONINE KINASES ARE AUXIN TRANSPORT AND ROOT GRAVITROPISM COMMON IN PLANTS Auxin levels are directed at the root tip for Receptor Serine/Threonine Kinases are the where epidermal growth occurs largest cell surface receptors in plants (rare When a root is directed 90 degrees, it in animals where RTKs and GPCRs are the responds to gravity by redirecting most common) downwards. Leucine-Rich Repeat (LRR) Receptor Auxin inhibits epidermal cell growth when kinases (175 LRRs in Arabidopsis thaliana) the root is directed laterally. Ligands include plant steroids such as brassino-steroids Bri1 cell-surface Receptor kinase – binding of brassino steroids Lectin Receptor Kinases – bind to carbohydrate signal molecules ETHYLENE SIGNALING PATHWAY Plant hormones such as ethelene, auxin, gibberillins, abscisic acid, and brassicosteroids regulate plant growth and development. Ethelene is a gas hormone that controls fruit ripening, leaf abscission, plant senescence, and response to stress (infection, flooding, etc.) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 LIGHT RESPONSE IN PLANT CELLS Photoproteins – light sensitive proteins to monitor the quantity, quality, direction and duration of light. Phytochromes – dimeric serine/ threonine kinases which respond to red and far-red light Cryptochromes – flavoproteins that detect blue light CYTOSKELETONS ARE DYNAMIC AND ADAPTABLE Cytoskeletal structures can change or persist depending on the cell’s needs (ranges from seconds to entire life span of a cell) THE CYTOSKELETON Component molecules of cytoskeletons are in constant state of flux. CYTOSKELETON Protein filaments responsible for the cell’s strength, shape, ability to move. Play spatial and mechanical functions. Types: ○ Actin Filaments ○ Microtubules ○ Intermediate filaments ACTIN FILAMENTS OR MICROFILAMENTS Helical polymers of actin protein 8 nm diameter arranged into linear bundles, 2D networks, or 3-dimentional gels Flexible structures dispersed around the cell but concentrated in the cell cortex beneath the plasma membrane. Functions: FUNCTIONS OF CYTOSKELETONS ○ Provide strength and shape of Actin filaments plasma membrane ○ Determine cell shape ○ Form cell surface projections ○ Cellular locomotion (lamellipodia and filopodia) ○ Cytokinesis ○ Attachment to substratum Microtubules (basement membrane) ○ Determine location of organelles ○ Muscle contraction ○ Direct intracellular transport ○ Tilting of rods in hair cells in inner ○ Forms mitotic spindles ear in response to sound Intermediate filaments ○ Increase surface area for absorption ○ Provide mechanical strength in microvilli ○ Active streaming of cytoplasm in plant cells Actin Filaments or Microfilaments RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ○ Forms nuclear lamina beneath the inner nuclear membrane serving as protective cage for DNA ○ Provides mechanical strength in the cytoplasm ○ Span the cytoplasm of one cell to the next in epithelial cells to provide strength to the entire epithelial tissue ○ Form long and robust axons in neurons ○ Form tough integumentary appendages such as hairs and nails. MICROTUBULES Long and hollow cylinders composed of tubulin proteins. 25 nm in diameter With one end attached to microtubule organizing center (MTOC) or centrosome. Functions: ○ Spindle fiber formation during cell division ○ Formation of cilia (locomotor organ RAPID REOGRANIZATION OF CYTOSKELETON and sensory device) Crawling movement of fibroblasts is due to ○ Formation of flagella (locomotor polarized dynamic actin cytoskeletons organ) (red) ○ Tracks for transport vesicles Polarization of cell is assisted by ○ Direct the pattern of cell wall microtubule cytoskeletons (green) synthesis in plants Microtubules form bipolar mitotic spindles ○ Form the cell framework in during cell division and pulls chromosomes protozoans. during anaphase Actin filaments form contractile ring during cytokinesis Microtubules and actin cytoskeletons reorganize the daughter cells INTERMEDIATE FILAMENTS Ropelike fibers with about 10 nm diameter Composed of large heterogenous family of intermediate filament proteins Functions: RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 CYTOSKELETON IN MITOSIS Cytoskeletons form multiple Cytoskeletons subunits assemble and protofilaments to become thermally disassemble regularly stable. Result of polymerization and Microtubules are composed of 13 depolymerization protofilaments arranged in circular helix formation. NEUTROPHILS CHASE BACTERIA THRU CYTOSKELETON REORGANIZATION Neutrophils engulf foreign bodies by forming protrusive structures filled with newly polymerized actin filaments. Rapid disassembly and reassembly of actin filaments. FTSZ FILAMENT: A BACTERIA TUBULIN HOMOLOG FtsZ Filaments are found in eubacteria and archea They polymerize to form a Z-ring at the site CYTOSKELETON IN POLARIZED EPITHELIAL of septum during cell division CELLS Highly dynamic filaments with half life of Cytoskeletons determine polarity (apical 30 seconds and basal surface) Site of enzymes needed in septum Actin form microvilli and circumferential formation band connects cell-cell adherens junctions Intermediate filaments are anchored in desmosomes and hemidesmosomes Microtubules run from top to bottom to provide global coordinate system and direct organelle movement. MREB AND MBL: BACTERIA ACTIN HOMOLOG Found in rod-shaped and spiral-shaped bacterial cells MULTIPLE PROTOFILAMENTS ARE THERMALLY Assemble to form dynamic patches that STABLE move circumferentially along the length of Cytoskeletons are composed of subunits the cell (actins and tubulins) Scaffold for the synthesis of peptidoglycan Subunits as connected by weak of bacterial cell walls non-covalent interactions (easy to Half-life: few minutes (highly dynamic) assemble and disassemble) Mutations of the MreB gene results to Cytoskeletons in single protofilaments are abnormal cell shapes and defects in thermally unstable chromosome segregation RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ○ β actin – expressed in non-muscle cells ○ 𝛾 actin – expressed in non-muscle cells Actin monomers are arranged into 2 protofilaments forming ParM AND ParR IN PLASMID SEGREGATION Actin homologs in bacteria Encoded by antibiotic resistance plasmids ParM assembles into filament that associate with each copy of plasmid ParR are DNA binding proteins that bind to the plasmid and stabilizes the dynamic ends of ParM. ParM separates the plasmid copies to each end. ACTIN FILAMENTS HAS TWO ENDS 2 Actin Filaments form filamentous or F-actin which is a right handed helix with 8 nm diameter. Minus end (pointed end) – slower growing Plus end (barbed end) – faster growing CRESCENTINS IN CAULOBACTERS ACTIN FILAMENT NUCLEATION Homologous to intermediate filaments Polymerization of actin subunits to form Crescentins have coiled coil domains actin filaments is important in determining forming filamentous structures responsible cell shape and movement. for the sickled shape of Caulobacter Subunits assemble spontaneously first as crescentus oligomers (lag phase) then as growing When crescentin gene is mutated, the cell filaments (growth phase). becomes straight. Equilibrium phase – when rate of polymerization balances the rate of depolymerization NUCLEOTIDE HYDROLYSIS Each actin molecule (monomer) has a tightly bound GTP ACTIN MONOMER AND PROTOFILAMENTS GTP is hydrolyzed to GDP during Actin subunit (monomer, globular or polymerization G-actin), 375 aa with tightly associated ATP Each monomer in the actin filament or ADP (polymer) has tightly bound GDP Highly conserved in eukaryotes (90% Critical Concentration – rate of subunit identical) addition is equal to rate of subunit loss 3 isoforms in vertebrates: ○ ⍺ actin – expressed in muscles RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ACCESSORY PROTEINS OF ACTINS More than a hundred actin binding proteins involved in different functions inside cells Binding to actin monomers: ○ Profilin ○ Arp2/3 Complex ○ Formin ○ Thymosin Binding to actin filaments: ○ Tropomodulin ○ Cofilin ○ Gelsolin ○ Fimbrin ○ Filamin ○ Capping protein ○ ERM ○ Tropomysin THREADMILLING ○ Alpha actinin Phenomenon when filament growth is achieved despite continuous polymerization (addition of monomers) and nucleotide hydrolysis (removal of monomers) Threadmilling occurs because the rate of addition in + end is faster than hydrolysis despite the catching up of hydrolysis to addition in the minus end. DYNAMIC INSTABILITY When individual microtubules alternate between a period of slow growth and a period of rapid disassembly. Microtubules depolymerize about 100 THYMOSIN AND PROFILIN times faster in an end containing the Regulate actin polymerization GDP-tubulin than from the end containing Thymosin binds to actin monomers to the GTP-tubulin inhibit polymerization on both plus and GTP cap favors growth but undergoes minus ends of actin filament depolymerization when lost. Profilin binds to actin monomers to promote polymerization on the plus end. Profilin activity is regulated by phosphorylation and binding to inositol phospholipids ACTIN AND MICROTUBULE INHIBITORS Application: ○ Prevents cell division (anti cancer drugs and chemotherapy) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 ACTIN WEB FORMATION Filament nucleation is the creation of an initial actin polymer (nucleus) to facilitate polymerization. Actin-nucleating proteins bring several actin monomers to form a seed (nucleus) ARP 2/3 (Actin related Proteins) – 45% homology with actins ARP2/3 complex nucleates actin filament growth from the minus end at 70º angle to existing filaments forming interconnected webs of actin filaments FILAMENT CAPPING Capping proteins bind at the end of filaments to regulate polymerization. CapZ – plus-end capping protein found in the Z-line of muscle cells, reduces filament growth and depolymerization. Arp2/3 complex – minus-end capping FILAMENT NUCLEATION VIA FORMINS protein Formins are dimeric actin nucleating Tropomodulin – minus-end capping proteins that nucleate growth of a straight, protein in long-lived actin filaments in unbranched filaments. muscles Formins remain bound to filaments at the plus end Many formins are indirectly connected to plasma membrane and aid insertional polymerization of actin filaments GELSOLIN AS ACTIN-SEVERING PROTEIN Gelsolins are activated by high levels of cytosolic Ca2+ Gelsolins bind to the sides of actin filaments PROFILIN ENHANCES FORMIN-NUCLEATION Thermal fluctuation cleaves the filament Some profilins contain unstructured and gelsolin serves as a capping protein at domains (whiskers) that bind to the plus end. profilin-actin complex. Whiskers serve as staging area for addition of actin at the plus end. Repeated reloading of actin using the whisker domain of formin results to elongation of the actin filament. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 COFILIN TWISTS AS ACTIN FILAMENTS Alternating plus and minus ends of Cofilin or Actin Depolymerizing Factor is a filaments included in the bundle 14 kDa protein binds along the length of the of the actin filament forcing the filament to twist more tightly. Preferential binding to ADP-bound actin filaments. Weakens the interactions of actin monomers resulting to brittle and easily severed by thermal motions (rapid disassembly) FILAIM CROSS-LINKING OF ACTIN Filamin homodimer (160 nm long) binds 2 actin filaments in a cross pattern. Forms a flexible high angle links of actins Mutation of filamin A gene causes periventricular heterotropia resulting to epilepsy. ACTIN FILAMENTS ARRAY IN FIBROBLASTS Different arrangements of actin filaments influence cellular mechanical properties and signaling. Formation of dendritic networks are nucleated by Arp2/3. Tight parallel bundles as caused by formins ACTIN-BASED MOVEMENT OF L. MONOCYTOGENES ACTIN CROSS-LINKING PROTEINS Listeria monocytogenes hijacks the host’s Fimbrin contains 2 adjacent actin binding cytoskeletons to move inside the host cell. sites, 14 nm apart ActA protein of bacteria activates the ⍺-Actinin with 2 actin binding sites Arp2/3 complex, capping protein and separated by 30 nm long spacer Filamin is cofilin to cause actin polymerization which V shaped protein with 2 actin-binding sites propels the bacteria at a rate of 1um/sec Spectrin is a tetramer (2 ⍺ and 2 β subunits) leaving a “comet tail” effect. with 2 actin binding sites separated by 200 nm distance ACTIN FILAMENTS BUNDLES ACTIN BINDS TO MYOSIN Bundling Proteins – actin binding proteins Myosin II is a motor protein (generates that form actin filament bundles. force for muscle contraction) Fimbrin cross-links filaments into tight 2 Heavy chains contain a globular head (N bundles terminus), neck or hinge region, and long ⍺-Actinin is a homodimer that loosely coiled-coil tail (C terminus) forming alpha binds filaments. helices RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 2 Light chains binding on the neck region of heavy chains. MYOSIN II BIPOLAR THICK FILAMENT MUSCLE Myosin II filaments form a bundle of filaments in muscle cells where tails interact with other tails in opposite directions. Myosin heads in a filament bundle are oriented on opposite directions. Bare zone contains tails only without heads MYOSIN HEADS HYDROLYZE ATP Each myosin head binds and hydrolyzes OPTICAL TRAP EXPERIMENT ATP where energy generated is used to A procedure to illustrate the movement of move along an actin filament towards its myosin along an actin filament plus end. The movement of the beads attached on Motor activity of myosin heads immobilized both ends of actin serves as optical tweezers in a glass slide was shown through the to monitor the movement. movement of actin filaments (red and Displacement is recorded over time. green) in the presence of ATP. Arrows indicate direction of actin filament movement. MYOSIN II WALK ALONG ACTIN Myosin II uses the conformational change SKELETAL MUSCLES in the ATP binding site to generate movement along actin filament (power Muscle bundle is composed on myofibrils stroke). which consists of repeated chain of ○ Myosin head interacts tightly with sarcomeres (contractile units) actin filament in the absence of ATP. Z-discs separates 1 sarcomere from ○ Binding of ATP to myosin head another. detaches the head from the actin Sarcomeres are composed of overlapping ○ ATP hydrolysis in the myosin head parallel myosins and actins releases phosphate generating force to move myosin head ○ Myosin head bound to ADP binds to actin about 8.5nm from original binding site towards to plus end. ○ Removal of ADP (causes power stroke) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 TROPONIN AND TROPOMYOSIN CONTROL MUSCLE CONTRACTION Control of muscle contraction is influenced by accessory proteins troponin and tropomyosin. Tropomyosin – binds along the groove of actin filaments Troponin T, I, and C (troponin complex) – binds to actin molecule Troponin T (tropomyosin-binding) and Troponin I (Inhibitory) – pulls tropomyosin which inhibits binding of myosin head to actin. Troponin C (Ca binding) – binds Ca2+ which causes troponin I to release actin SARCOMERE ORGANIZATION Overlapping thick (myosin) and thin (actin) filaments Muscle contraction is sarcomere shortening caused by sliding of myosin heads over the actin filaments. Plus end of actin is capped by CapZ interacting with ⍺-actinin to form Z disc. Minus end of actin is capped by tropomodulin Nebulin binds along the entire length of the actin filament SMOOTH MUSCLE CONTRACTION Titin controls sarcomere length during Absence of striations in smooth muscles is muscle contraction and relaxation by caused by irregular arrangement of binding to Z disc to the M Line. contractile proteins Contraction depends on calmodulin (absence of troponins) Calmodulin bound to Ca2+ activates Myosin Light Chain Kinase (MLCK) Phosphorylation of Myosin Light Chain by MLCK results to smooth muscle contraction INCREASE OF CA2+ INITIATES MUSCLE CONTRACTION T-tubules extends inward from the plasma membrane around each myofibril Ca2+ channels on the membranes of T-tubes and sarcoplasmic reticulum control Ca2+ release into the cytoplasm. Sudden increase in cytosolic Ca2+ concentration initiates muscle contraction. MUTATIONS IN CARDIAC MUSCLE PROTEINS Familial Hypertrophic cardiomyopathy – genetic condition associated with heart enlargement, small coronary vessels, and disturbances in heart rhythm (cardiac arrythmias) RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Caused by point mutations in the genes MYOSIN V CARRIES CARGO ALONG ACTINS coding for cardiac β myosin heavy chain or Myosin V is a two-headed myosin with mutations on the genes for myosin light large step size for movement of cargo along chains, cardiac troponin, and tropomyosin actin filaments. Missense mutation in cardiac actin gene Involved in portioning of organelles such results to dilated cardiomyopathy (early as peroxisomes and mitochondria heart failure) MICROTUBULES Polymers of tubulin protein composed of a heterodimer of ⍺ and β subunits with 445-450 aa each connected by noncovalent bonds. ⍺ subunit binds GTP that cannot be hydrolyzed or exchanged because it it ACTIN AND MYOSIN FUNCTIONS IN trapped in the dimer interface. NON-MUSCLE CELLS β subunit binds GTP that can be hydrolyzed Regulated by myosin phosphorylation by to GDP MLCK which forms self assembly of bipolar Monomers form protofilament with plus myosin filament bundle (15-20 filaments) and minus ends Formation of cortical stress fibers that 13 protofilaments arranged in helical connects cells to extracellular matrix via conformation form a microtubule with focal adhesions, formation of hallow lumen. circumferential belt, or adherens junctions MYOSIN SUPERFAMILY Common N terminus motor domain and variable C terminus One-headed or two-headed myosin molecules Found in both plant and animal cells (early evolution) 40 myosin genes in humans MICROTUBULES HAVE + AND - ENDS 9 human myosins in hair cells of inner ear ⍺-tubulins are exposed at the minus end (mutations resulting to hereditary β-tubulins are exposed at the plus end deafness) Both ends can be used for polymerization Some myosins are involved in microvilli and depolymerization but the plus end movement and endocytosis grow and shrink more rapidly. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 DYNAMIC INSTABILITY OF MICROTUBULES Rapid interconversion between growing (polymerization) and shrinking (depolymerization) at a uniform subunit concentration. Catastrophe – change from growth to shrinkage Rescue – change from shrinkage to growth CENTROSOMES IN ANIMAL CELLS GTP hydrolysis – favors depolymerization Centrosomes contain the MTOC of animal GTP cap – favors polymerization cells positioned near the nucleus. Microtubules are nucleated at centrosomes where their minus ends are connected to centrioles while plus ends point outward for growth and shrinkage A PAIR OF CENTRIOLES IN CENTROSOMES Centrioles are cylindrical modified microtubules arranged in right angles against each other (L-shaped configuration) with nine-fold symmetry of microtubule triplets. Centrosome duplicates and splits before cell division. DYNAMIC INSTABILITY OF MICROTUBULES Migration to opposite poles of dividing cell Newt epithelial lung cells undergoing generates orientation of chromosome microtubule growth and shrinkage movement (polarity of the cell) INHIBITORS OF MICROTUBULE POLYMERIZATION AND DEPOLYMERIZATION Polymer-stabilizing and polymer-destabilizing drugs preferentially kill dividing cells (since microtubule dynamic instability is crucial in the MICROTUBULE ARRAY CAN FIND THE CENTER OF functions of mitotic spindles) THE CELLS Used as chemotherapeutic drugs Microtubes influence the shape of the cells (drawback: unspecific cytotoxicity to both by forming star-shaped formation from cancer and non cancer cells) centrosome to sides of the cell. Absence of centrosomes results to star-shaped microtubules assembly at the center of the cell with the minus end clustered at the center and plus ends extending to the sides of the cell, MICROTUBULE ORGANIZING CENTER Nucleation (assembly) of microtubules starts at MTOC 𝛾-tubulin ring complex (𝛾TuRC) – initiates nucleation of microtubules together with accessory proteins Serves as a template for 13 protofilaments to polymerize as microtubule polymer RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 MICROTUBULE-ASSOCIATED PROTEINS Phosphorylation by kinases of MAP2 and 𝛾-Tubulin Ring Complex Tau protein control their microtubule Stathmins binding activity and localization in the cell. Kinesins Katanins Plectins XMAP215 MAP2 Tau +TIPs (Plus end Tracking Proteins) Microtubule-Associated Proteins) MAPS ON MICROTUBULE ENDS Kinesin 13 (Catastrophe factor) binds at the plus ends to promote depolymerization of microtubules (shrinkage) XMAP215 – stabilizes the plus ends and promotes polymerization by binding to free tubulin dimers and bringing them to plus ends (growth) Phosphorylation of XMAP215 inhibits its activity. Nezha or Patronin prevents catastrophe at MICROTUBULE the minus end of microtubules Tau proteins (green) bind to microtubules into bundles along the axons of hippocampal neuron MAP2 proteins (red) bind to microtubules within the cell body and dendrites of hippocampal neuron PLUS END TRACKING PROTEINS +TIP EB1 protein is expressed in growing microtubule (frames 1 and 2) but not expressed during catastrophe or shrinkage (frames 3 and 4). It is expressed again when rescue takes place (frame 5). MAP2 AND TAU FORM MICROTUBULE BUNDLES MAP2 form bundles of microtubules that are widely spaced because of its long projecting domains. STATHMINS SEQUESTERS TUBULINS Tau protein form more compact bundles of Stathmin proteins (Op18) binds to two microtubules because of its short projecting tubulin heterodimers and prevent them domain. from being used for polymerization. RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 Stathmin decreases the concentration of MOVEMENT OF KINESIN ALONG MICROTUBULE tubulin monomers available for growth of The 2 kinesin motor domains work in a tubulins coordinated manner – 1st motor protein in Phosphorylation of stathmin prevents its front and the 2nd in the rear end. binding to tubulins. The leading head binds ADP (loose binding microtubule) while the lagging head binds ATP (tight binding to microtubule). During “kinesin step” ATP bound to lagging head is hydrolyzed creating a movement to the next microtubule binding site. Removal of ADP to be replaced by ATP on the original leading head. Movement towards the plus end of microtubule (anterograde movement) KATANINS SEVERE MICROTUBULES Severing microtubules is a means to destabilize microtubules (disassembly). Katanins has 2 subunits – large and small subunits Small subunit of Katanin severe microtubules by hydrolysis of ATP (bound to tubulins) Large subunit of Katanin releases microtubules from the MTOC which contributes to rapid microtubule depolymerization during cell division. DYNEIN MOTOR PROTEINS Movement of vesicles towards the minus end of microtubules (retrograde movement) With 1, 2 or 3 heavy chains, intermediate chain and light chain Types: ○ Cytoplasmic Dynein Cytoplasmic Dynein 1 – organelle and mRNA KINESIN MOTOR PROTEINS trafficking Kinesin 1 – used for vesicle transport. With Cytoplasmic Dynein 2 – two heavy chains - N terminus forming intra-flagellar transport (tip globular head and long tail in coiled coil to base of cilia or flagella) conformation, two light chains that ○ Axonemal Dynein (ciliary dynein) – interact with cargo vesicle. heterodimers or heterotrimers Motor protein domain – common to all kinesin proteins RIVERA, JADE BEATRICE - CELL AND MOLECULAR BIOLOGY LECTURE FINALS| FIRST TERM| ACADEMIC YEAR 2024-2025 POWER STROKE OF DYNEIN Large protein with 4000 aa Motor head contains 6 AAA domains (1 major ATPase) and C-terminal domain Stalk is detached from microtubule but re-attaches during ATP hydrolysis. Release of ADP and Pi produces “power stroke” of about 8 nm involving rotation of the motor head and stalk relative to the tail DYNACTIN HELPS DYNEIN IN TRANSPORT Dynactin mediates the attachment of MICROTUBULES IN CILIA AND FLAGELLA cytoplasmic dynein to a membrane