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Multicellularity: From cells to tissues to organisms ECB 16-1 shmoos Mating dance of a budding yeast (S.cerevisiae)… Haploid a- and a-cells form shmoos in response to chemical signals Shmoos mate to form diploid a/a cell Examples of: - “differentiated” cell types (a-, a-, and a/a-cells) cell-cell adhesion -cell-cell signaling Human body consists of trillions of cells, 200+ specialized cell types that must differentiate (next time) and communicate (today) with one another Cell-cell communication required to coordinate: - physiology and metabolism - behavior -growth, proliferation, and differentiation Basic categories of cell-cell signaling in animals “Contact-mediated” (short range) “Paracrine” (local) ex. - nerve cell production ex. inflammation Signaling cell Signaling cell Target cell Target cells “Autocrine” “Endocrine” (long distance) “Neuronal” (ex.-hormones) Endocrine (signaling) cell Post-synaptic target (muscle, neuron, etc) Target cells Axon hormone Action potential Cell body of neuron Bloodstream Synapse ECB 16-3 Cellular response depends on specific combination of signals ECB 16-6 No signal often results in activation of apoptosis Common features of cell-cell signaling pathways Other signals ECB 16-7 Receptors for diffusible signals can be intracellular or surface Cell surface receptors Intracellular receptors Small non-polar molecules cross plasma membrane by simple diffusion Large polar molecules …cannot cross membrane They bind cell surface receptors ECB 16-9 And bind to intracellular receptors Intracellular signals Plasma membrane Transcription Transcription Most receptors for hydrophobic signaling molecules act in nucleus to regulate gene transcription Membrane receptors for hydrophilic signaling molecules activate a wide variety of intracellular “signal transduction” pathways, including gene regulation A few examples of hydrophobic hormones ECB 16-11 CH2OH OH C=O HO O OH OH O HO Cortisol Estradiol Testosterone I HO HO O I Cholesterol (not hormone) I H CH2 I C COO- NH3+ Thyroid hormone Responses mediated by a conserved family of “steroid” receptors Responses to hydrophobic hormones are mediated by intracellular receptors Plasma membrane Target cell Cytoplasm Intracellular receptor Translation Nuclear envelope Lipophilic hormone carried in blood Hormone binds intracellular receptor inducing receptor dimerization and activation Complex is imported into nucleus Binds to “hormone response element” to regulate gene expression Transcription “Hormone response element” Promoter Target gene Nucleus ECB 16-12 Cell-surface receptors - three classes Ion channel-linked receptor Signaling ligand Ions G-protein linked receptor Target Signaling ligand Target (inactive) Receptor G-protein (inactive) Target (inactive) Receptor G-protein (active) (active) (active) Receptor G-protein (active) (active) Enzyme-linked receptor Signaling ligand Catalytic domain (active) Catalytic domain (active) ECB 16-14 Activation of surface receptor can cause fast (cytoplasmic) or slow (transciptional) changes Review: phosphorylation and GTPases as molecular switches Signaling with phosphorylation Signal in Signaling with GTPases Signaling protein Signal in Off Off Pi ATP Signaling GTPase GDP Pi GDP Kinase Signal activates protein kinase Phosphatase GEF Signal activates GEF GTP ADP On On P Signal out GTP ECB 16-15 Signal out Energy (in the form of ATP or GTP hydrolysis) used to activate (or inactivate) signaling molecules Energy use allows transient, high affinity/specificity interactions GAP “Heterotrimeric G-proteins” mediate many cell signals b g Ga, Gbg subunits a Ga binds guanine nucleotide GDP GDP Gabg (inactive GDP form) Heterotrimeric G-proteins Pi Receptor acts as GEF, activating G-protein Activated Ga- and Gbg regulate targets Ga inactivated by GTP hydrolysis, subunits reassociate GTP Active Ga and Gbg (GTP form) b g See ECB 16-17 + a GTP Downstream targets Multiple G-proteins with distinct a-, b-, and g-subunits (>20 known) “Gs” stimulates or activates effectors “Gi” inhibits effectors “Gq” mediates Ca2+ signaling “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors G-protein –GDP Ligand binding domain (inactive) Extracellular space b g 1 2 3 4 5 a6 7 Plasma membrane Inactive receptor GDP Cytoplasm Effector domain See ECB 16-16 Seven transmembrane domains (a-helices) Extracellular ligand-binding domain (N-terminal) Cytoplasmic “effector” domain Activated receptor acts as GEF to activate “heterotrimeric G-protein” “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors G-protein –GDP (inactive) b g Seven-pass receptor a GDP Inactive target Binding of ligand activates receptor ECB 16-18 thru 16-18 “Heterotrimeric G-proteins” are activated by a family of “Seven-pass” transmembrane receptors b g a Active receptor GTP GTP GDP Binding of ligand activates receptor Heterotrimeric G-protein binds activated receptor Activated target ECB 16-18 thru 16-18 Activated receptor acts as GEF for heterotrimeric G-protein Activated components (a- and b/g-) regulate downstream targets GTP hydrolysis inactivates G-protein, subunits reassociate (switches off) Activated target can be enzyme that makes “intracellular messenger” ECB 16-20 Ephinephrine (adrenaline) acts via heterotrimeric G protein and cAMP (intracellular messenger) Activated adenylate cyclase forms cAMP cAMP activates protein kinase A (PKA) PKA enters nucleus and phosphorylates a gene regulatory protein ECB 16-24 Result: altered transcription (slow) Adenylate cyclase converts ATP to 5’,3’ cAMP O A O O 5’ - O P O P O P O CH2 - O - O - O O 4’ 3’ 2’ OH OH “Adenylate cyclase” PPi A 5’ CH2 O O P - O 3’ 2’ O OH O O 4’ Adenosine 3’,5’ cyclic monophosphate (cAMP) A 5’ - 2Pi Methylated xanthines (caffiene, theophylline, and theobromine ) inhibit cAMP PDE O P O CH2 O 1’ O cAMP phosphodiesterase - ATP 1’ 3’ OH 1’ 2’ OH AMP ECB 16-21 cAMP levels rise rapidly in response to extracellular signal Assay fluorescence of protein that binds cAMP 5 X 10 ECB 16-22 -8 M cAMP 10 -6 M cAMP Serotonin is a neurotransmitter G-protein coupled receptors also activate IP3 and Ca2+-mediated signaling pathways Activate receptor acts as GEF Activated Ga activates phospholipase C (PLC) Active PLC cleaves PIP2 to IP3 and diacylglycerol (DAG) IP3 opens Ca2+ channels in ER releasing Ca2+ to cytoplasm DAG and Ca2+ activate protein kinase C (PKC) Active PKC phosphorylates target proteins… Other Ca2+-dependent responses are regulated by “Calmodulin” (CaM) and “CaM kinases” Ca2+ CaM contains 4 Ca2+ binding domains Inhibitory domain Catalytic domain Inactive CaM kinase Ca2+ Ca2+-CaM binds to regulatory domains of effector proteins (e.g. CaM kinases) ADP ATP P Autophosphorylation Ca2+ Calmodulin (CaM) Active CaM kinase Phosphorylates target proteins in cytoplasm see ECB 16-27 Ca2+-calmodulin activates CaM kinases, which phosphorylate and regulate target proteins Cells carefully regulate “free” Ca2+ levels in their cytoplasm Ca2+ [Ca2+] >1 mM ATP ADP + Pi Ca2+ 2Na+ In a resting cell, intracellular [Ca2+]free is low relative to external Ca2+… Ca2+ is pumped into the ER (plant vacuole) [Ca2+ ]free ~0.2 mM ATP ADP + Pi Ca2+ ER Ca2+ is pumped out of the cell by a Ca2+ ATPase and antiport with Na+ (antiport with H+ in plants/fungi) Intracellular [Ca2+]free may increase 10-30-fold during signaling… Moves in through channels and is released from internal stores (mostly from the ER, vacuole) Last class of cell surface receptors Signaling ligand 1. Ligand gated ion channel Ions 2. G-protein coupled receptor Target Signaling ligand Target (inactive) Receptor G-protein (inactive) Receptor G-protein (active) (active) 3. Enzyme-linked receptor Signaling ligand Catalytic domain (active) Target (inactive) Catalytic domain (active) (active) Receptor G-protein (active) (active) Many growth factors bind to receptor tyrosine kinases (enzyme-linked receptor) Receptor binds growth factor and dimerizes Kinase activity activated and receptor autophosphorylates Signaling proteins bind phosphotyrosine, activating signaling cascades EGF and other growth factors activate Ras signaling Ras found to be mutated in ~30% of human tumors! RAS (inactive) GTP Exchange Factors (GEFs) promote GDP/GTP exchange GDP Pi “Off” GDP GAP GEF GTP “On” “GTPase Activating Protein” (Ras-GAPs) promote GTP hydrolysis by intrinsic GTPase RAS Active Ras activates downstream signaling proteins… GTP Downstream effectors Receptor tyrosine kinases activate intracellular Ras signaling cascades Growth Factors P RAS P RAS MAPKKK (inactive) GTP P P P P DRK GDP inactive active MAP kinase kinase kinase (MAPKKK) Ras GEF Receptor kinase (active) ATP ADP P MAP kinase kinase (MAPKK) MAPKK MAPKK active inactive ATP ADP Downstream of Receptor Kinase activates Ras GEF P “Mitogen-activ. protein kinase” MAP kinase P MAP kinase inactive active ATP ADP P ECB 16-31, 16-32 Transcription factors P Other proteins Regulate gene expression and protein activity Mutations in Ras signaling pathway cause uncontrolled cell proliferation: cancer P RAS P RAS MAPKKK (inactive) GDP P P P P DRK active GTP ATP ADP Ras GEF P GTP MAPKK active Receptor kinase (active) ATP ADP Downstream of Receptor Kinase The Ras pathway activates expression of G1 cyclins that stimulate cell proliferation P P MAP kinase active ATP Constituitive activation of pathway components results in uncontrolled cell proliferation = “cancer” Cancer causing genes = “Oncogenes” Predict effects of Ras mutations? ADP P Transcription factors P Other proteins Regulate gene expression and protein activity… Signal transduction cascades are complex and interconnected G-protein coupled receptors G-protein G-protein P P P P P P Phospholipase C IP3 Adenylate cyclase Diacylglycerol Receptor tyrosine kinases • Integration Adapter Ras activator Ras Ca2+ cAMP Calmodulin Protein kinase A CaM kinase Gene regulatory proteins Protein kinase C Why? Multiple inputs to a single response… • Divergence Single input to multiple responses Kinase I • Amplification Kinase II • Regulation Kinase III Cytoplasmic target proteins ECB 16-38 Communication by direct cytoplasmic continuity between cells Cytoplasmic bridges and cell junctions Communication via cell junctions: some embryonic cells and/or tissues are “dye-coupled” 100 Da 1,000 Da 10,000 Da Membrane-impermeant dye injected into on cell passes into neighbors Cytoplasmic coupling is limited to small molecules (<1000 Da) “Gap junctions” are responsible for cytoplasmic coupling of animal cells Membranes of coupled cells closely apposed, separated by 2-4 nm “gap” Large “gap jnctn” TEM/Freeze fracture of gap junctions reveals “plaques” of intra-membrane particles ECB figure 19-28 MBoC figure 19-16 Common in developing embryo, cardiac muscle, liver, and lens Gap junctions are composed of “connexons” made of “connexin” hexamers Cytoplasm of cell #1 Channel is ~ 1.5 nm (~1000 Da cutoff) Plasma membrane of cell #1 “Connexon” (2 per channel) = “connexin” x 6 Extracellular “gap” (2-4 nm) Plasma membrane of cell #2 Cytoplasm of cell #2 Two connexons in register form channel coupling cytoplasm of adjacent cells ECB 21-28 The cytoplasm of plant cells is coupled by “plasmadesmata” Cytoplasm Desmotubule Cell wall Cytoplasm Vacuole Nucleus Plasma membrane of adjacent cells Cell wall Nucleus Cytoplasm Nucleus Plasmadesmata Endoplasmic reticulum 100 nm ECB 21-30 Membranes continuous from cell to cell ER continuous from cell to cell thru “desmotubule” Limited to small molecules (<800 Da), but can open to let through 20,000 Da Primarily (but not exclusively) formed during cell division