BIOL311 Exam 3 PDF

CHAPTER 12: CELLULAR ENERGETICS Mitochondria Structure Outer membrane Inner membrane Intermembrane space Matrix Inner Mitochondrial Membrane has 3 parts 1. Boundary membranes: Flat membrane next to the outer membrane 2. Cristae: Folding and tube-like invaginations of...

CHAPTER 12: CELLULAR ENERGETICS Mitochondria Structure Outer membrane Inner membrane Intermembrane space Matrix Inner Mitochondrial Membrane has 3 parts 1. Boundary membranes: Flat membrane next to the outer membrane 2. Cristae: Folding and tube-like invaginations of the inner membrane 3. Cristia junction: sharp bends that connect the cristae with the boundary membrane Mitochondrial membranes Outer membrane Has porins that allow small molecules through when they are open Inner membrane Contain proteins for oxidative phosphorylation These proteins include: ○ ATP synapse ○ Electron transport proteins ○ Transport proteins These membranes create Intermembrane space Space between the inner and outer membrane Continuous with the lumen of the cristae Matrix Central compartment inside the inner membrane Mitochondrial DNA (mtDNA) Located in the matrix Circular DNA (like bacterial DNA) mtDNA codes for  ○ Several proteins essential for mitochondrial function (Tim, some electron transport proteins) ○ Ribosomes ○ Transfer RNAs (tRNA) Most proteins in the mitochondrial are codes for the cell nuclear DNA ○ Are made in the cytosol and are transported into the mitochondria Mitochondria are constantly changing Mitochondria are tubular and branched network Dynamic ○ Continue to change Mitochondria show rapid fusion and fission Fusion When inner and outer membrane of two mitochondria fuse to make one Why do they do this? To keeps mitochondria homogenous ○ Allows mitochondria to be mostly the same within the cell Fission Fragments off damaged or mutated mitochondrial parts ○ Parts are then removed by autophagy Fragments mitochondria for: ○ Easily separate them into dividing cells ○ Easily moves them to parts of the cell that need them Production of ATP Adenosine triphosphate ○ Adenine, ribose, 3 phosphates Sites for the production of ATP from ADP + inorganic phosphate from glucose ○ Minimal amount from glycolysis ○ Mainly by aerobic respiration by the mitochondria ○ Also by photosynthesis by chloroplasts Has 3 high energy phosphate bonds Which creates energy currency of the cell NAD (Nicotinamide adenine dinucleotide) Another energy molecule in the cell Carries 2 electrons (and a proton) Used in oxidation/reduction reactions Glucose 6 carbon sugar Key to ATP production Glucose breakdown pathway is central to: Catabolic pathways: break things down Anabolic pathways: build things into larger things Glycolysis is the breakdown of glucose into 2 pyruvate Take 2 pyruvates into acetyl CoA Acetyl CoA will feed into citric acid cycle Carbohydrates are broken down into sugars, fats and phospholipids can be broken down into glycerol and fatty acids, proteins can be broken down into amino acids with release of NH3, which can be made into glucose and then follows into pathway Glucose can be put together into glycogen (branched, polymers of glucose and storage protein in animals) or starch Glycolysis can make substrates for nucleotide synthesis Can take acetyl CoA to make fatty acids such as phospholipids and fats Take from citric acid cycle to make substrates for amino acid synthesis Essential for cells metabolism Glucose oxidation Glucose oxidation takes place in 4 stages 1. Glycolysis Occurs in the cytosol, so is not a mitochondrial function Produces only small amount of ATP and NADH Pyruvates are 3 carbon sugars 2. Citric acid cycle Occurs in mitochondrial matrix 2 pyruvates so everything is doubled Produces NADH, FADH2 and some ATP Results Glycolysis: 2 ATPs (or GTPs) 2 NADH Citric acid cycle (from conversion of 2 pyruvates) 2 ATPs 6 NADH 2 FADH2 (+2 NADH from oxidation of pyruvates → acetyl CoA) 4 ATPS + 10 total NADH + 2 FADH2 3. Electron transport chain Embedded in the mitochondrial inner membrane ○ Complexes I, II, III and IV Uses NADH and FADH2 to produce the proton-motive force ○ Electro-chemical gradient of H+ across the inner mitochondrial membrane into the intermembrane space 4. ATP synthesis Requires the ATP synthase enzyme Uses the proton-motive force to phosphorylate ADP to ATP ATP synthase Multiprotein complex in the mitochondrial inner membrane Uses energy of the proton-motive force to phosphorylate ADP to ATP ○ As ATP synthase allows protons (H+) to move back into the matrix ○ ATP synthase causes phosphorylation of ADP to ATP Overall result Approximately 30 ATP are made from one glucose CHAPTER 15: SIGNAL TRANSDUCTION: GPCR AND ADENYLYL CYCLASE Cell-to-cell communication By: Hormones Pheromones Some neurotransmitters Immunology cytokines  ○ Cytokine are a generic term for signaling molecules involved in immunology Requires: A signal that can be transmitted by: A soluble factor released from one cell or a group of cells ○ Steroid hormones, growth, inflammatory factors, a cytokine, etc. A receptor on the target cell for that signal ○ Cell membrane protein receptor or cytosolic receptor for the factor OR by direct cell-cell interactions Cell with a membrane protein ligand Binds to a membrane protein receptor on another nearby cell But in BOTH cases, the signal only affects cells that have the receptor ○ No receptor on the cell= no effect on the cell Types of extracellular/intercellular signaling A. Endocrine signaling a. Signal secrete by a cell b. Enter the blood c. Travels to a distant site d. Binds to receptors on target cells i. Hormones like insulin, epinephrine, etc. B. Paracrine signaling a. Signal secreted by a cell b. Bind to cell surface receptors on nearby cells i. Neurotransmitters, growth factors, T lymphocytes activating B lymphocytes, etc. C. Autocrine signaling a. Cells secretes factors that acts on itself b. Self stimulatory i. Growth factors ii. Problem with cancers cells stimulating their own growth D. Signaling by plasma-membrane attached proteins a. Requires a direct cell-to cell contact b. Interaction between cell surface receptors c. 1 to 1 or 1 to a few Effect Endocrine and paracrine May be one or a few cell affecting many target cells Autocrine and paracrine Often act together NADH has more potential energy and enters the the electron transport chain (at Complex II) above FADH2 (Complex III) so more protons are transported across the membrane. Discussion Questions: 1. Why does NADH yield more proteins transported across the inner mitochondrial membrane than FADH2? It feeds into all the complexes. NADH has more energy. 2. Which type of ATP-powered pump is the ATP synthase classed as? F-class pumps Characteristics of signaling receptors Two major groups of receptors 1. Receptors inside the cell Bind to hydrophobic signaling molecules ○ Can diffuse across the plasma membrane without the need of a transport protein ○ Many are steroids, vitamin D, thyroid hormones, etc. ○ The receptor is in the cytosol, nucleus or other organelles Steroid hormones Cholesterol derivatives Hydrophobic signaling molecules Diffuse across the plasma membrane ○ No surface receptor Hydrophobic signal diffuses across the plasma membrane into the cytosol Binds to a receptor in the cytosol Receptor changes conformation  Becomes an active Transcription Factor to alter gene expression 2. Hydrophilic signals binding to cell surface receptors Hydrophilic signals Extracellular signaling molecules: ○ Small molecules, like epinephrine, acetylcholine, etc. ○ Peptides, like glucagon (29 amino acids) ○ And proteins like insulin, growth hormone, etc. Bind to cell surface receptors Usually integral membrane protein Induces a conformational change in the receptor ○ Transmitted through the transmembrane domain to the cytosolic domain Receptor binds to and activates other proteins into the cytosol to tranduce the signal into the cell Membrane receptors eventually: 1. Activate enzymes 2. Induces changes in the cytoskeleton 3. Activate transcription factors that turn on or turn off genes Cell surface receptors Most receptors bind only a single type of ligand ○ Receptor are very specific But several different receptors many bind a single type of ligand ○ May have the same effect or a different effect on different cells ○ Acetylcholine Induces Skeletal muscles to contract Heart muscles reduce the rate of contraction Pancreatic cell to secrete digestive enzymes ○ Bind to ligand with high affinity Kinases and phosphatases Cell surface receptors usually result in the activation of kinases and/or phosphatases Kinases ○ Enzymes that add phosphate groups to specific amino acids on target proteins  ○ Cause conformational change in the target protein Due to the negative charged phosphate That will activate target protein ○ Kinases add phosphate groups to: Tyrosine amino acids Serine amino acids Threonine amino acids Phosphatases Enzymes that remove phosphate groups from specific amino acids on target proteins Returns the protein to its native conformation Generally: Kinases add phosphates to activate proteins Phosphatases remove phosphate to inactive proteins BUT THIS IS NOT ALWAYS TRUE! ○ Kinases can add phosphates to inactivate some proteins ○ Phosphatases can remove phosphates to activate some proteins Many receptors activate or inactivate g-proteins Two types of g-proteins 1. Monomeric g-proteins (or small GTPases) Molecular switches ○ +GTP=On ○ Hydrolyzes GTP to GDP= off Examples: ○ Ran ○ Sar1 and ARF ○ Rab ○ Rho, Cdc42, Rac 2. Large heterotrimeric G-proteins (hetero=different subunits; trimeric= 3 parts) Have 3 parts: GB and Gy subunit ○ Always associate together  ○ Have a lipid tail that embeds into the membrane to bring it close to the receptor Ga subunit ○ Has a lipid tail to bring it to the membrane ○ Can dissociate and associate with GBy ○ Binds to GDP when resting ○ Binds to GTP when activated ○ If it has an s it a stimulatory Trimeric G-protein Activated by cell surface receptors called: G-protein coupled receptors (GPCR) Humans have about 800 different GPCRs GPCRs A. Contain 7 transmembrane alpha-helical regions B-Adrenergic receptor for epinephrine ○ Controls smooth muscle contraction Serotonin receptor ○ In the CNS controls depression/anxiety Histamine H1 receptors ○ Induces smooth muscle contraction in allergic responses B. Associates with a heterotrimeric g-protein 1. Acts as an on-off switch activated by the GPCR Binding the ligand (hormone, etc.) Induces a conformational change in the GPCR This activates the Trimeric G-protein  ○ GPCR acts as the guanine nucleotide exchange factor (GEF) ○ Induces exchange of GDP for GTP C. Activated trimeric g-protein alpha subunit: Ga subunit undergoes a conformational change ○ Due to the bound GTP Ga subunit separates from the GBY subunits Ga subunit interacts with membrane-bound effector to activate it Downstream effector proteins They do the effect of the signaling 1. Membrane ion channel proteins Gated channels that open to allow ions out of the cell K+ channels 2. Membrane bound enzymes Adenylyl cyclase Phospholipase C Etc. Alter metabolism, cell movement, turn on certain genes Regulation of trimeric g-protein signaling 3 mechanisms: 1. GTP is spontaneously and rapidly hydrolyzed to GDP by the Gα subunit Gα rapidly returns to off conformation Gα then associates with the GBy subunits 2. GTP hydrolysis can be enhanced by a GTPase-activating protein (GAP) Results in an even faster inactivation Seconds to minutes signal duration Regulation of trimeric g-proteins (cont.) 3. Hormone-induced inhibition of the effector protein Other hormones may activate an inhibitory GPCR This inhibitory GPCR activates inhibitory GPCR Activated by Gαi inhibitory subunit turns off the effector protein Effector proteins Many effector enzymes associated with GPCRs induces the production of second messengers (first messenger are the original signaling molecule/hormone) Second messengers are molecules or ions that bind to and activate proteins in the cell This activated proteins that cause physiological changes in the cell Effector enzymes can rapidly produce high levels of second messengers to amplify the effect 3’,5’-cyclic adenosine monophosphate (cAMP) 1,2- diacylglycerol (DAG) Inositol 1,4,5-triphosphate (IP3) GPCR that activate adenylyl cyclase Adenylyl cyclase is an effector protein Transmembrane protein Activated adenylyl cyclase produces cyclic AMP ○ From ATP Example: low blood sugar levels Epinephrine and glucagon hormones in the blood Activate GPCR on liver cells Activates a stimulatory trimeric G protein Stimulatory Gαs activates adenylyl cyclase Production of high levels of cAMP Results in breakdown of storage glycogen to increase glucose in the cell Adenylyl cyclase can have dual regulation But prostaglandin E1 and adenosine Bind to different GPCR on liver cells Activate inhibitory Gαi Results in No cAMP so no breakdown of glycogen What does cyclic AMP do? Activated adenylyl cyclase produces MANY cAMPs cAMP activates many protein kinase A Amplifies the effect Protein kinase A (PKA) Tetramer of: 2 regulatory subunits 2 catalytic subunits  Activation of PKA 4 cAMP bind to sites on the regulatory subunits Regulatory subunits release the 2 catalytic subunits The catalytic subunits are now active Protein kinase A active catalytic subunits Serine/threonine kinase ○ Add phosphate groups to serine and threonine amino acids of the target proteins PKA function ○ Inhibit enzymes for glycogen synthesis ○ Activates enzymes for glycogen degradation These increase glucose levels in the cell PKA activity stimulates gene transcription 1. Activated PKA enters the nucleus 2. PKA phosphorylates the CREB transcription factor 3. CREB then turns on many genes in glucose synthesis Discussion Questions 1. How can a cell inhibit trimeric g-protein function? Spontaneous breakdown of GTP to GDP, GAP protein activation, hormone induces an inhibitory g-alpha 2. Why would the cell want to rapidly downregulate the activation of a trimeric g-protein? Important for regulation 3. Activation of protein kinase A can have an immediate effect and a delayed effect. What are these two effects?  Protein kinase A phosphorylates serine and therones and activates effector enzymes in the cytosol. Second affect: turning on of genes in glucose synthesis CHAPTER 16: SIGNAL TRADUCTION: RECEPTOR TYROSINE KINASES Brief gene regulation Promoter= regulatory region of the gene ○ Usually before the gene ○ Site where RNA polymerase binds ○ Has the start site for transcription Transcription factors ○ Bind to the DNA ○ Turn on/off transcription ○ One or more may be involved in regulation Signal transduction Transmits the activating signal into the cell Binding the ligand to the receptor activates signal transduction pathways within the cell ○ Turn on enzymes ○ Induce changes in the cytoskeleton, metabolism, etc. ○ Activates transcription factors that turn on genes Many different signal transduction pathways in the cell Ligand binds to the receptor Activated receptor leads to activating a specific signaling pathway We will focus on the receptor tyrosine kinase pathway Receptor tyrosine kinases (RTKs) Epidermal growth factor receptor Fibroblast growth factor receptor Nerve growth factor receptor Insulin receptor Many others Receptor tyrosine kinase pathway Common type of signaling for many protein hormone and growth factors The receptor has 4 parts ○ External ligand-binding site (and dimerization arm) ○ Transmembrane portion ○ Protein tyrosine kinase domain Have an activation loop ○ Tail end with several tyrosine that can be phosphorylated 4 human epidermal growth factor receptors (HER) HER 1 ○ Binds to human epidermal growth factor ○ Also binds to 6 other membrane of the EGF family  TGF-a and neuregulins and others ○ Require a homodimer (two HER1s) for signaling HER 4 ○ Binds to several EGF family ligands ○ Requires a homodimer (two HER4s) for signaling HER 2 ○ CANNOT bind any ligand! ○ But it can form a functional heterodimer with HER 1, HER 3, or HER 4 ○ So Dimerization arm is always open So it can dimerize with a HER that has bound a ligand So it can form heterodimers and transmit the signal But homodimers are not functional The tyrosine kinase domain is functional There is no HER2 HER2 HER 3 ○ Can bind to a ligand ○ But does not have a functional kinase domain ○ It can work with HER 2 ○ HER 3 binds the ligand and then dimerizes with HER 2 (which has a functional kinase domain) to form a functional unit Functional dimers BINDS EGF FUNCT. HOMO-DIM HETERO- KINASE ERS DIMER with HER 2 HER 1 + + + + HER 4 + + + + HER 3 + - - +  HER 2 - + - - HER 3 only functions with HER 2 HER 2 cannot make homodimers HER 2 can make heterodimers with HER 1,3, and 4 Many cancers overexpress HER 2 Enhances signaling with other HER receptors Important target for cancer therapy Gefitinib (Product name: Iressa) ○ Inhibitor that can inhibit HER signaling Transtuzumab (Herceptin) ○ Inhibits signaling through HER 2 Activation of RTKS Unstimulated RTK Usually a monomer in the plasma membrane Kinase activation loop NOT phosphorylated ○ Loop blocks the enzyme active site ○ Inactive kinase Activation of RTKs– ligand binding Binding of the ligand causes interaction of dimerization arms of the receptors Receptor dimerization brings the 2 intracellular kinase domains together Activation of the tyrosine kinases Interaction of the 2 kinase domains: Causes a conformational change in the kinase domains This pushes out the activation loop from one of the kinases This kinase is now active This kinase then autophosphorylates several tyrosines on the activation loop of itself and the second kinase These 2 kinases are now fully activated The activated kinases recognize short sequences on the cytosolic tails of the receptors that contain tyrosine amino acids They then autophosphorylate these tyrosines on the cytosolic tails of the receptors to create phosphotyrosine sequence sites These sequences with a phosphotyrosine are now a binding site for other proteins Other proteins have SH2 domains ○ This is a binding domain on the protein ○ It recognizes short amino acid sequences with the phosphorylated tyrosine ○ SH2 domains can bind to phosphotyrosines This is how the cell can get 2 different proteins to bind to each other Proteins with SH2 domains phosphotyrosines on other proteins Downstream signaling of RTKs Many RTKs signal by activating the small GTPase Ras ○ EGF, PDGF, FGF, etc. These all activate small GTPase ras Ras then activates MAP Kinase signal transduction pathway Activation of Ras via GRB2 and Sos Ras needs to be linked to cytosolic tail of the activated RTK GRB2 Adapter protein Has no enzymatic activity Only serves to link two proteins together Has an SH2 Domain And two SH3 domains ○ SH3 domains bind to proline-rich sequence in other proteins Sos Inactive small GTPase Ras needs a Guanine Nucleotide Exchange Factor (GEF) Sos is GEF that induces Ras to exchange its GDP for GTP ○ Ras-GTP is active So Sos must be linked to the activated RTK and Ras GRB2 Adapter protein functions to link sos to the RTK ○ Sos has the Proline-Rich sequences that the SH3 domains on GRB2 can bind Binding sos activates Ras Binding of Sos to GRB2 brings Sos to the membrane Now Sos can bind to inactive Ras-GDP ○ Ras is bound to the membrane by a lipid link Sos is a GEF that can induce Ras to exchange its GDP for GTP Ras-GTP is now active Ras-GTP then activates the MAP Kinase signal transduction pathway ○ While Sos-GRB2-RTK can then activate another Ras The MAP kinase pathway The MAPK pathway is a 3 part signaling pathway Ras activates a MAP3K (MAP Kinase Kinase Kinase) MAP3K phosphorylates to activate MAP2K MAP2K phosphorylates to active MAPK MAPK does the effect Raf is the MAP3K Raf is inactive because: 1. Phosphorylated at multiple sites at the kinase enzyme domain a. Here phosphorylated is turned OFF 2. And bound to the inhibitory protein 14-3-3 Ras-GTP activation of Raf Ras binds to the autoinhibitory domain of raf Induces a conformational change in Raf This leads to the removal of 14-3-3 Ras then hydrolyzes its GTP to GDP Ras is released from the Raf Two Rafs dimerize The Raf then phosphorylate each other Rafs become fully active Activate Raf phosphorylates MEK MEK is the MAP2K Active Raf phosphorylates MEK MEK becomes an active kinase Active MEK then phosphorylates MAP kinase Map kinase becomes an active kinase The MAP kinase Pathway *There are several signaling pathways similar to this sequence Why have this signaling pathway? Amplifies the response ○ GPCR and trimeric G-proteins amplify by the production of many second messengers ○ RTK signals get amplified by activating Many Ras Activate Raf Each Raf can activate many MEK Each MEK can activate many MAP kinase Function of MAP kinase Phosphorylate and activate several transcription factors that turn on gene ○ C-Fos transcription factor Activate the Early Response Genes Genes for proteins important in: ○ Proliferation, cell survival, cell differentiation How do we get these signaling proteins together? Scaffold proteins  ○ MAP Kinase pathway Scaffold protein is KSR ○ Brings Raf, MEK and MAP kinase (ERK) together ○ Insures that signaling will occur Regulation of RTK signaling 1. Ras is rapidly inactivated by hydrolysis of GTP 2. Active MAP Kinase (ERK) can phosphorylate Raf to inhibit Raf from binding to KSR 3. MAP Kinase (ERK) activates Transcription Factors that produce phosphatases a. Phosphatase dephosphorylate MAP Kinase Discussion questions 1. Kinases can phosphorylate tyrosine amino acids on proteins. Give 2 functions for these phosphorylated tyrosines they can become binding sites for SH2 domains for other proteins and cause conformational changes 2. We have specific chemical inhibitors that can inhibit function of specific kinases and many of these are used as drugs for various diseases a. What would be an important limitation for using these specific kinase inhibitors? b. What might be a better target to inhibit a signaling pathway? Inhibit the scaffold protein CHAPTER 20: EXTRACELLULAR MATRIX AND CELL JUNCTIONS Cell and tissues Many cell types White blood cells (leukocytes) Red blood cells (erythrocytes) Fat cells (adipocytes) Many subtypes of neurons Fibroblasts Etc. Tissues  Cells of various types that aggregates to cooperatively perform a common function ○ Epithelial tissue: compartmentalize, selective barrier, etc. ○ Connective tissue: support ○ Muscular tissue: contraction ○ Neural tissue: conduct electrical impulses ○ Blood: transport Organs Different tissues organized together to perform one or more function Made of several tissues together ○ Intestines ○ Muscles ○ Heart ○ Etc. A major tissue type: Epithelia Tightly packed sheets of cell that allow: Compartmentalization and selectively permeable barrier ○ Lining of stomach ○ Skin ○ Linking of the intestine, etc. Also includes endothelium lining inside of blood vessels (single layer of cells) Principle types of epithelia Simple columnar ○ Elongated cells ○ Example: secretion and absorption cells of intestine Simple squamous ○ Thin flat cell ○ Line blood vessels (endothelium) or body cavities Transitional ○ Layers of cells that can expand and contract (bladder) Stratified squamous ○ Line surfaces of mouth and vagina Cell-adhesion molecules (CAMs) Cell surface receptors that bind to cell surface receptors on neighboring cells Cell to cell interactions ○ Including specialized cell junctions Connect cells together Allow communication between cells to coordinate functions Cell matrix adhesion Cell surface adhesion receptors that bind to extracellular matrix Extracellular matrix: complex of proteins and polysaccharides that surround the cells Cell adhesion molecules 4 major families 1. Selectins a. Have a lectin domain on the tip end i. Lectin: binds to specific sugars on glycoproteins or glycolipids b. Relatively weak interactions c. Major function in leukocyte binding to endothelial cells i. Helps them enter tissues from the blood at sites of inflammation 2. Immunoglobulin (Ig) superfamily a. Have repeating immunoglobulin domains i. Similar to binding sites of antibodies b. Strong binding c. Bind to either i. Other Ig superfamily CAMs on other cells ii. Some bind to integrin CAMs 3. Integrins a. Made of an alpha (a) and a beta subunit b. 8 types of B subunits c. 18 types of a subunits i. Come together to produce 24 different integrins  d. Mediate i. Cell-cell interactions 1. (a1B2 binding to Ig superfamily CAM (ICAM-1) ii. Cell-ECM interactions 1. (a5B1 binding to fibronectin) Subunit Primary Cellular Distribution Ligands Composition α1β1 Many types Mainly collagens α2β1 Many types Mainly collagens; also laminins α3β1 Many types Laminins α4β1 Hematopoietic cells Fibronectin; VCAM-1 α5β1 Fibroblasts Fibronectin α6β1 Many types Laminins αLβ2 T lymphocytes ICAM-1,1CAM-2 αMβ2 Monocytes Serum proteins (e.g., C3b, fibrinogen, factor X); ICAM-1 αIIbβ3 Platelets Serum proteins (e.g., fibrinogen, von Willebrand factor, vitronectin); fibronectin α6β4 Epithelial cells Laminin B1 family binds to ECM proteins Many cells types express integrins Several integrins bind to the same ECM protein (redundant) VCAM, ICAM-1, ICAM-2 are Ig superfamily CAMS Many integrins link to intracellular signaling pathways Some can activate the MAP kinase pathway ○ Induce proliferation Some link to Rho, Rac and Cdc24 ○ Signaling for actin polymerization and cell movement There is a cross-talk between integrin signaling and RTK signaling ○ Cross regulation of each other ○ RTK signaling can turn on/off some integrins 4. Cadherins a. Cell-cell recognition and communication receptors b. Over 100 different cadherins c. 6 different families d. Brain expresses the largest number of cadherins i. Necessary to establish complex wiring patterns Classical cadherins E,N, and P-cadherins (epithelial, neural and placental) Found in adherens junctions between cells and along sides next to other cells Require calcium to bind ○ Hence the name: calcium adhering= cadherins Cadherins structure Have 5 extracellular domains ○ EC1-EC5 The EC1 domain can bind to EC1 domains on ○ Cadherin on a nearby cell= trans interaction ○ Cadherin on the SAME cell= cis interaction Cadherins first bind by trans (intercellular) interactions EC1 domain of cell bind to EC1 domain of cell 2 ○ This is a trans interaction between two different types of cells Then they bind cis interactions Trans interactions increase the probability of cis interactions occurring EC1 domains of nearby cadherins on the SAME cell then bind to each other Strengthens the bond between the cell Trans and cis interactions Trans interactions: between EC1 of cadherin on cell 1 to EC1 of cadherin on cell 2 Cis interactions: between EC1 of cadherin on cell 1 to adjacent EC2 on cell 1 Cadherins mediate cell-cell binding Cell labeled with GFP-E-cadherin Within 2 hours= start to see fluorescence at cell-cell interaction Fluorescence increases with time Suggests “zipping up” of the membranes together by cis interactions Cadherin cytoplasmic tails interaction with the cytoskeleton Cytosolic tails of cadherins bind to: B-catenins B-catenins then bind to: A-catenin Links to the actin cytoskeleton Gives the cell shape and stress information Cadherins may play a role in cancer spreading Epithelial cell conversion into malignant carcinoma cells Highly mobile cells Low expression of cadherins Many cell types are polarized Their plasma membrane are organized into discrete domains Neurons Simple columnar epithelial cells Migrating cells Polarized epithelial cells Apical (top) surface Basal (bottom) surface Lateral (side) surface Many have microvilli on the apical surface ○ Fingerlike projections of the membrane ○ Increase absorption area Attached to the ECM at the basal surface ○ Basal lamina Cell-cell and cell-ECM junctions There are many individual CAM-mediated adhesions between cells But epithelia have specialized clusters of CAMs ○ Mediate cell-cell and cell-ECM interactions ○ Give strength and rigidity to tissues ○ Control movement of molecules and ions across epithelia ○ Transmit information between extracellular and intracellular spaces And form tight junctions between the cells 3 types of cell-cell junctions Anchoring junctions  ○ Hold tissues together Tight junctions ○ Control the passage of ions and molecules through the extracellular space between cells Gap junctions ○ Serve as conduits for the movement of ions and molecules from the cytoplasm of one cell to its neighbor A. Anchoring junctions 1. Adherens junction 2. Desmosomes 3. Hemidesmosome 4. Focal adhesions 1. Adherens junction Connect lateral membranes of adjacent cells Principle CAM are cadherins Connect to actin filaments (adherens belt) on the inside of cells Along with the belt of actin and myosin filaments that circle the apical area of the cell: ○ Brace the cell and control its shape 2. Desmosomes like “snaps” that hold the membranes of adjacent cell together Made of desmosomal cadherins Interact with intermediate filaments Function in giving strength and rigidity to the cell 3. Hemidesmosomes Found on the basal surface Bind the cell to the ECM Principle CAM are a6B4 integrins Attach to intermediate filaments Give shape and rigidity to the cell 4. Focal adhesions Discrete cell-ECM interactions Integrin binding to various ECM proteins Dynamic structures Important interactions with: ○ Actin microfilaments ○ Cell movement ○ Signaling pathways ○ Sensing the environment Mostly integrin extracellular matrix interactions Discussion questions: 1. What is the immunoglobulin superfamily of receptors? Cell adhesion molecule that binds to other immunoglobulin superfamily and sometimes integrins. The immunoglobulin domains are the binding sites. 2. Why have “cross-talk” between integrin signaling pathways and RTK signaling pathways Allows for cross-regulation of each other. RTK can turn on and off the integrins. B. Tight junctions Found mainly in tissues that seal off body cavities ○ Epithelia in the intestine, bladder, etc. ○ Endothelia of blood vessels Form barriers that seal off cavities–water tight Prevent movement of water ions, macromolecules across epithelia Membrane of adjacent cells are “stitched together like a quilt” Proteins of the tight junction Occludin  ○ Function to seal the TJs Claudins ○ Many types of these (27) ○ Some seal and some allow some molecules through Junction adhesion molecules (JAM) Tight junctions prevent movement of molecules across epithelia between cells Experiment: Shows tight junction preventing movement of substance between cells So molecules cannot leak from the basal → apical or apical → basal surfaces Movement of molecules across epithelia Paracellular transport Movement through channels in the tight junction Most movement of molecules between cells is tightly closed The cell can change claudins to allow some selective movements between the cells ○ Size and ion selective Transcellular transport Endocytosis of the molecules on the basal side Transport of the endocytic vesicle across the cell Exocytosis of the molecules from the apical side C. Gap Junctions Tubes that form channels through the membrane of adjacent cells Composed of connexin proteins that create a hexagonal complex The channels let through things that are 1200 daltons or less ○ Ions ○ 2nd messengers ○ Even some nutrients from metabolism Function of gap junctions Allow sharing of signals between adjacent cells to coordinate multicellular events Allow cells to rapidly pass ionic signals among cardiac muscle to coordinate heart beating Coordinate peristaltic muscular contraction in the intestine The extracellular matrix Complex of proteins and polysaccharides Secreted by the cells Assembled by the cells Very dynamic ○ “Remodeled” by chemical modification and proteolysis Purpose: To hold cells and tissues together ○ But much, much more Dense and sparse ECM Connective tissue can have lots of ECM Epithelial cells may have very little ECM ○ Mostly in the basal lamina beneath the epithelial cells The basal lamina Thin sheet-like meshwork Beneath epithelial cells Or surround other cells Function of the basal lamina Provide an anchor for epithelial cell binding Organize cells into tissues or compartments Role as a scaffold in tissue repair  Forms the blood-brain barrier A selectively permeable blood filter for the kidney Types of ECM components Lamnin Large (~820 kDa size) Cross shapes Made of 3 chains ○ One each of 5 alphas, 3 betas and 3 gamma chains ○ Form 16 different laminin isoforms ○ Laminin 111 (a1 B1 y1) ○ Laminin 511 (a1 B1 y1) ○ They do different functions and are found in different places Laminins structure Ends of the cross structure have multiple binding sites For self (other laminins) For integrins ○ Laminin is a major binding protein for a cell surface integrins to bind to the basal lamina For collagen LG domains bind ○ Cell receptors ○ Other ECM glycoprotein Collagens (type of ECM components) ~28 different types ○ Collagen 1= tendon and skin ○ Collagen IV= basal lamina Made of: ○ Collagen alpha chain ○ Then 3 collagen a chain twist into a triple helix Creates long fibrils Collagen type IV Has triple helix fiber with occasional non-helical segments ○ Allows kinks in the fiber  Also has a large globular end Collagen IV can bind to other Collagen IV ○ Head to head ○ Tail to tail ○ Allows it to form sheets when they bind to each other Perlecan A proteoglycan ○ Protein (proteo) with attached chains of sugars (glycans) Long protein chain With attached long glycosaminoglycans ○ GAGs are long polymers of repeating disaccharides Binds to laminin and other ECM components ○ Cross-links larger ECM proteins together Nidogen (Entactin) Binds to: ○ Collagen IV ○ Perlecan ○ Laminin ○ Glue that stabilizes the basal lamina Composition of the basal lamina 1. The plasma membrane of the cells have integrins that bind to the laminin sheet 2. Laminins bind together to form sheets 3. Sheets are cross-linked by Nidogen, Perlecan and Laminin 4. Then there is a collagen IV layer that binds to the Laminin sheet via the laminins and nidogens Loose connective tissue Loose collagen fibers ○ Produced by fibroblasts Elastic fibers of Elastin ○ In tissues subject to mechanical strain ○ Blood vessels, lungs, skin Glycosaminoglycans (GAGs) ○ Heparin sulfate, chondroitin sulfate, hyaluronan ○ Attract water to lubricate and act as shock absorbers Dense connective tissue Tendon ○ Mostly fibrillar collagens ○ Produced by fibroblasts ○ Function is for strength Fibronectin Produced by many cell types A multi-adhesion ECM protein Usually dimers bound together by disulfide bonds Having binding sites for ○ Heparin sulfate (a GAG) ○ Collagen ○ Integrins/cells ○ Fibrin in clots Function of fibronectin Essential for movement of many cell types ○ Bind to integrin/cell binding sites Bind cells to the ECM ○ Cell integrin binds to FN → FN binds to the ECM Allows for cross-links in the ECM Essential for wound healing ○ Fibrin binding site allows cells to move over clots Matrix metalloproteinases (MMP) Proteases that can degrade ECM proteins ○ Collagenases, elastases, etc. Function in remodeling the ECM ○ Morphogenesis in development Removal of ECM to help sculpt body parts ○ Wound repair Remove clots and etc. for repair of the ECM and tissue ○ Cancer cells produce MMPs Allow spreading of cancer to other parts ot eh body Metastasis Discussion questions 1. What type of cell-cell junctions prevent paracellular movement of molecules?  Tight junctions→ claudin, occludin and JAM 2. If an epithelia cannot do paracellular transport, how does a molecule or protein (ions, food sugars, etc.) get across the epithelial layer? Transcellular transport 3. What is needed for cells at the edge of a wounded epithelium to move into/across the wound to heal the wound? Matrix metalloproteinases, need fibronectin CHAPTER 19: MITOSIS AND THE EUKARYOTIC CELL CYCLE Eukaryotic cell cycle Process that includes: Growth in cell size Duplication of cellular organelles DNA replication Chromosome separation (mitosis) Separation of organelles, cytoplasm, and plasma membrane between the two daughter cells Has 4 major phases Mitosis Mitosis M phase Stage when replicated chromosomes are separated into the daughter cells Interphase Contains 3 phases (plus G0) ○ G1= preparing for DNA synthesis ○ S=DNA synthesis ○ G2 preparing for mitosis How to determine cell cycle? Isolate cells Stain DNA with fluorescent dye Use a flow cytometer to determine fluorescent content of the cells 1C amount of DNA ○ Cells in G1/G0 phase 2C amount of DNA  ○ Cells in G2 and M phase Area between 1C and 2C ○ Cells actively replicating DNA ○ S phase Cell cycle Overall takes about 24 hours ○ G1 phase ~9 hours ○ S phase ~ 10 hours ○ G2 phase ~4.5 hours ○ M phase ~30 minutes G0 phase Cell is outside of the cell cycle ○ Non dividing Phase where the cell does what it does ○ Liver cell function ○ Muscle cells in normal muscle ○ Neurons transmitting signals ○ Etc. Is reversible in many cell types ○ Cell can re-enter cell cycle and beginning cell division G1 phase Gap 1 phase Starts after mitosis is completed Cell has 2n set of chromosomes: ○ For humans ○ n=23 different pairs of chromosomes ○ 2n=46 total chromosomes G1 phase Cell must evaluate: Cell size ○ Is big enough to be separated into 2 cells Nutrient status ○ Are there enough nutrients to warrant more cells? Substrate attachment  ○ IS it attached to ECM Density of neighboring cells ○ Is there enough room for more cells? Presence of growth factors and chemicals that stimulate cell division ○ Is the cell being told to undergo cell division How does the cell assess these? Obtains information from signaling pathways that are attached to Integrin signals from the ECM There are other signaling pathways for information Mitogens: ○ Growth factors or chemicals that promote cells to go through cell cycle and divide ○ They are signals that activate RTK signaling pathways ○ Such as EGF, FGF, etc. ○ Bacterial wall components for immune cells Cell cycle checkpoints Found at the border between different cell cycle phases Restriction point (G1-S checkpoint) Monitor cell growth G2-M checkpoint Consists of sensors that monitor ○ Growth, DNA replication Restriction point (G1-S checkpoint) late in G1 phase When cells have enough size, nutrients, etc. Cell can pass the restriction point Point in G1 phase Once passed the restriction point, the cell is committed to cell division