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  1. 1. Lecture 36: Membranes and Membrane Proteins 11/29/2011 1:34:00 PM Cell membranes act as selective barriers Prevent molecules from mixing Three roles of plasma membrane Receiving information (signaling) Import/export (transportation) Motility/cell growth Membranes enclose many different compartments in a eukaryotic cell: Nucleus (2x) Mitochondria (2x) ER, vesicles, golgi apparatus, lysosome, peroxisome The Lipid Bilayer Two-dimensional fluid Fluidity depends on composition Lipid bilayer is asymmetrical Lipid asymmetry is generated inside the cell Hydrophilic head, hydrophobic tail The more unsaturated the tails are, fluidity is increased Phosphatidylcholine is the most common phospholipid in cell membranes There are different types of membrane lipids and all are amphipathic Hydrophilic molecules attract water, like dissolves in like Hydrophobic molecules avoid water Fats are hydrophobic, phospholipids are amphipathic (and form a bilayer in water) Pure phospholipids can form closed, spherical liposomes Phospholipids can move o Lateral o Flexion o Rotation o Flip-flop Fluidity depends on composition o Cholesterol stiffens o Low temperature, less unsaturation, long tails all reduce fluidity. Phospho and glycolipids are distributed asymmetrically in the plasma membrane
  2. 2. o Glycolipids found on outside o Phosphatidyl-serine, inositol, ethanolamine found on inside usually.Flippases transfer phospholipids to other side of membraneNew membranes are synthesized from ER. o Form vesicles which fuse with other membranes Membrane ProteinsPolypeptide chain usually crosses the bilayer as an α-helixProteins can be solubilized in detergents and purifiedPlasma membrane is reinforced by the cell cortexCell surface is coated with carbohydrateCells can restrict movement of membrane proteinsFunctions o Transporters (ie Na pump) o Anchors (integrins) o Receptors (platelet-derived growth factor receptor) o Enzymes (adenylcyclase)50% of mass of plasma membranes, 50 times more lipid than proteinmolecules.Different ways of associating with membrane: o Alpha helix, beta pleated sheets, transmembrane, lipid linked o Can be peripheral (protein attached)Folded up proteins traverse membrane easier because the polarbackbone is exposedMultiple alpha helixes form a hydrophilic porePorin proteins form water-filled channels in the outer membrane of abacterium o Formed by 16 strands of β-sheets o Allows passage of ions and nutrients across outer membranes of some bacteria and of mitochondriaMembranes are disrupted by detergents such as SDS and Triton X-100 o Have only one tailBacteriorhodopsin acts as a proton pump powered by light, drives ATPsynthase
  3. 3. Plasma membrane reinforced by cell cortex – imparts shape andfunction o Spectrin meshwork forms the cell cortex in red blood cellsEukaryotic cells are sugar coated o Absorb water for lubrication o Cell-cell recognition o Protect cell from physical, chemical, enzymatic damage o Recognition of cell surface carb on neutrophils mediates migration in infectionMovement can be restricted by cells o Tethering to cell cortex, extracellular matrix, proteins on surface of another cell, or by barriers of diffusion like tight junctions.
  4. 4. Lecture 37I. General Principles of Cell Signaling Can act over long or short range Each cell responds to limited set of signals Signals relayed via intracellular signaling pathways Nitric oxide crosses plasma membrane and activates intracellular enzymes directly Some hormones cross plasma membrane and bind to intracellular receptors There are three classes of cell surface receptors Ion channel-linked receptors convert chemical into electrical signals Intracellular signaling proteins act as molecular switches Origins in unicellular organisms o Yeast shows single cell-cell communication o Two mating types, a and α plus a secreted mating factor signal Signals transduction: conversion of one type of signal into another o Extracellular -> intracellular 4 ways animal cells signal o Endocrine o Paracrine o Neuronal o Contact-dependent  Lateral inhibition: Unspecified epithelial cells, one cell is dedicated to becoming a nerve cell and inhibits surrounding cells by Delta-Notch signaling. One signal molecule can induce different responses in different cells o ie: acetylcholine: (time scale is seconds to minutes)  In heart muscle cells, causes decreased rate and force of contraction.  In salivary gland cells, causes secretion.  In skeletal muscle cells, causes contraction. An animal cell depends on multiple extracellular signals Extracellular signal molecules can alter activity of diverse cell proteins which in turn alter cellular behavior
  5. 5. o The intracellular signaling proteins are involved in a signaling cascade which ultimately reach the target proteins for altered behavior like metabolism, gene expression, and cell shape or movement.Cellular signaling cascades can follow a complex path o Primary transduction, relay, amplification, or branching to different targets.Extracellular signal molecules can either bind to cell surface receptorsor to intracellular enzymes or receptors (like nitric oxide) o Nitric oxide is a product of nitroglycerin which is taken to relax smooth muscle cells.  Triggers smooth muscle relaxation in blood-vessel wallSteroid hormones bind intracellular receptors that act as generegulatory proteins o Cross plasma membrane, like NO o Cholesterol does not cross membrane, rather inserts IN membrane. o Cortisol acts by activating a gene regulatory proteinMost signal molecules bind to receptor proteins on the target cellsurface o Extracellular domains are the cell surface receptor o Three basic classes:  Ion channel linked -> nervous system, muscle  G-protein linked -> all cells  Enzyme-linked -> all cellsMany intracellular signaling proteins act as molecular switches o Signaling by phosphorylation  Signal in by phosphorylation, off by phosphatase inactivation. o Signaling by GTP-binding protein  GTP binds to G-protein, turning it on.  GTP hydrolysis inactivates by removing P.II. G-protein-linked ReceptorsStimulation of G-protein linked receptorsG proteins can regulate ion channels
  6. 6. G proteins can activate membrane bound enzymesCyclic AMP pathway can activate downstream genesInositol phospholipid pathway triggers rise in CaCa signal triggers many biological processesIntracellular signaling cascades can achieve astonishing speed (iephotoreceptors in the eye)All G-protein linked receptors possess a similar structure o 7 transmembrane protein o Ligand binds to extracellular binding domain o Cytoplasmic domain which binds to G-protein o Tetramer is active, GDP can dissociate, GTP can bind, and then complex dissociates into two activated parts. o The alpha subunit switches itself off by hydrolyzing bound GTPG proteins couple receptor activation to opening of cardiomyocyte Kchannels o Acetylcholine binds to G protein linked receptor o Beta gamma complex binds to closed K channel to open it o Alpha subunit is inactivated (by hydrolysis) and inactive complex reassociates with betta gamma complex to close K channel.Enzymes activated by G proteins catalyze synthesis of intracellularsecond messengers o Alpha subunit activates adenylyl cyclase which makes lots of cylic AMP. o Cyclic AMP concentration rises rapidly in response to neurotransmitter serotonin o Cyclic AMP is synthesized by adenylyl cyclase, degraded by cAMPphosphodiesteraseExtracellular signals can act rapidly or slowlyRise in intracellular cyclic AMP can activate gene transcription throughprotein kinase A o Translocates through nuclear pore, into nucleus, phosphorylates gene regulatory protein to activate target gene.
  7. 7. Membrane bound phospholipase C activates two small messengermolecules: IP3, DAG o Phospholipase C activated by alpha subunit, splits inositol phospholipid into IP3 and DAG o IP3 opens Ca channel in ER, Ca is released and works with DAG to activate Protein Kinase C.Fertilization of an egg by sperm triggers a rapid increase in cytosolic Ca o Other processes triggered by Ca signal:  Sperm entry -> embryonic development  Skeletal muscle -> contraction  Nerve cells -> secretionCalcium/Calmodulin complex are what bind to proteins.A rod photoreceptor cell from the retina is exquisitely sensitive to light o G protein linked light receptor activates G protein transducing, activated alpha subunit causes Na channels to close. o Light induced signaling cascade in rod photoreceptors greatly amplifies light signals. III. Enzyme linked receptorsActivated receptor tyrosine kinases assemble a complex of intracellularsignaling proteins o Ligand brings two tyrosine kinase domains together, phosphorylated to activate. Intracellular signaling proteins bind to phosphorylated tyrosines. o Activated complex includes Ras-activating protein, which is anchored in membrane, transmits signal downstream.  Ras is monomeric GTP-binding protein, not a trimeric G protein, but resembles the alpha subunit and functions as a molecular switch.  30% of cancers arise from mutations in Ras.  Ras activates a MAP-kinase phosphorylation cascadeSome enzyme-linked receptors activate a fast track to the nucleusProtein kinase networks integrate info to control complex cell behaviorsMulticellularity and cell communication evolved independently in plantsand animals
  8. 8. Cytokine receptors are associated with cytoplasmic tyrosine kinases o JAK kinases phosphorylate receptor which recruits cytoplasmic proteins.TGF-beta/BMP receptors activate gene regulatory proteins directly atthe plasma membraneSignaling pathways can be highly interconnected: cross-talkLecture 38 General introduction:Membrane enclosed organelles are distributed throughout thecytoplasm o Thousands of different reactions occur simultaneously, are partitioned o Cytosol is 54% of cell o Mitochondria is 22% of cells o ER is 12% of cell (1 per cell)Nuclear membrane and ER may have evolved at the same time throughinvagination of plasma membrane.Mitochondria are thought to have originated from aerobic prokaryotebeing engulfed by a larger anaerobic eukaryotic cell -> has it’s owngenome.Nucleus is a double membrane organelle o Encloses nuclear DNA, defines nuclear compartment and contains most of the genetic information. o Export, import through nuclear pore complex  Contains about 100 proteins, two way gate, export of mRNA, and ribosome subunits.  Import of proteins requires a signal sequence called the nuclear localization signal  Requires energy (GTP) and special chaperone proteins  Export of RNA from nucleus – RNA molecules are made in the nucleus and exported to the cytoplasm as processed mRNAOne Endoplasmic Reticulum
  9. 9. o System of interconnected sacs and tubes of membrane o Extend throughout most of cell o Major site of new membrane (lipid) synthesis o With ribosomes on cytosolic side = rough ER o Without ribosomes = smooth ER o Most extensive network membrane in eukaryotic cellsGolgi apparatus o Flattened sacs called cisternae which are piled like stacks of plates o Usually near nucleus o Two faces:  Cis face adjacent to ER  Trans face towards plasma membrane (where post translational modification occurs) o Receives proteins and lipids o Site of modification of proteins and lipids o Dispatches proteins and lipids to final destinations o Transport vesicles bud offOther membrane enclosed organelles o Endosomes – small membrane enclosed organelles that sort ingested molecules in endocytosed materials. Passed to lysosomes or recycled back to the plasma membrane. o Lysosomes – small sacs containing digestive enzymes that degrade organelles, macromolecules, and particles taken in by endocytosis. “garbage disposal of the cell.” Ph about 7.2 o Peroxisomes – small membrane enclosed organelle containing oxidative enzymes that break down lipids and destroy toxic moleculesProtein transport o Multiple modes of protein transport (import and export) o Three mechanisms  Transport through nuclear pores: protein with nuclear localization signal enter through pores  Across membranes: proteins moving from cytosol into ER, mitochondria and peroxisomes transported across organelle membrane by protein translocators
  10. 10.  By vesicles: from ER onward and from one endomembrane compartment to another ferried by transport vesiclesProtein sorting signals o Specific amino acid sequence o Directs protein to organelle o Proteins without signals remain in cytosol o Signal sequences direct proteins to different compoartments  Continuous stretch of AA usually 15-20 residues in length  Usually removed after the protein reaches destination  Organelles and signal sequences:  ER import rich in V A L I and retendtion KDEL  Mitochondria rich in R  Nucleus PPKKKRKV  Peroxisomes SKL o Signal sequences are both necessary and sufficient to direct protein to organellesER: entry point for protein distribution o Proteins destined for golgi, lysosomes, endosomes and cell surfaces first enter ER from cytosol o Once inside ER or membrane, proteins do not reenter cytosol o Water soluble proteins are completely translocated across ER membrane and released into ER lumen o Transmembrane proteins only partially translocated across ER membrane and become embeddedVesicular transport o Entry into ER o To golgi apparatus o From er ->golgi -> other by continuous budding, fusion of transport vesicles o Vesicle transport provides routes of communicationProtein transport: quality control o Most proteins that enter ER are destined for other locations
  11. 11. o Exit from the ER is highly selective: improperly modified and or folded proteins are retained in lumen; dimeric or multimeric proteins that fail to assemble are also retainedExocytosis o Constitutive: newly synthesized proteins, lipids, and carbs delivered from ER via golgi to subcellular locations, extracellularly to ECM via transport vesicles.  Lipids and proteins supplied to plasma membrane  Proteins secreted into ECM or onto the cell surface o Regulated  Specialized secretory cells synthesize high levels of proteins such as hormones or digestive enzymes that are stored in secretory vesicles for subsequent release  Vesicles bud off from trans golgi network and accumulate adjacent to plasma membrane until mobilized by extracellular signalEndocytosis o Pinocytosis (drinking)  Internalizes plasma membrane: as much membrane is added to cell surface by exocytosis as is removed by endocytosis – total surface area and volume remain unchanged.  Mainly carried out by transport vesicles: deliver extracellular fluid and solutes to endosomes; fluid intake is balanced by fluid loss during exocytosis o Phagocytosis (eating)  Specialized cells only
  12. 12. o
  13. 13. Lecture 39: CytoskeletonRoles of cytoskeletal filaments: Intermediate – cell structure against mechanical stress Microtubules – intracellular transport, railroad of cell Actin – membrane mobility; cell movement Intermediate filaments: 10 nm in diameter Rope like structure composed of long polypeptides twisted together Associated with cell junctions Mechanical strength, cell shape, cell-cell contacts, and structure for nuclear envelope Monomers -> dimer -> tetramer -> 8 tetramers make one ropelike filament Different proteins: o Epithelia – keratins o Connective tissue, muscles, neuroglial cells – vimentin o Nerve cells – neurofilaments o Nuclear envelope in animal cells – nuclear lamins Mutation in keratin genes = epidermolysisbullosa simplex Networks of filaments connect across desmosomes in epithelia Microtubules: 25 nm wide Hollow, made of α and β tubulin anchored to γ tubulin Have polarity – gives directionality “Dynamic instability”: built or disassembled as needed o Zip up to grow o Unravel and tubulin molecules fall off if not needed o This is done by GTP since tubulin are GTPases  GTPases are the cell’s timers  High energy phosphate bond. Molecules with GTPases hydrolyze that bond, leaving GDP  GTP between α and β tubulin molecules makes them straighter, so they pack better. GTP hydrolysis makes them kinked, so they fall off.
  14. 14. Organize cell organelles and control traffic of vesiclesRoles in interphase cell, dividing cell, ciliated cells, flagella.The centrosome o Centriles inside of centrosome, nobody knows what they do o Centrosome is an envelope of tubulin where microtubules extend out with plus end out.Structure: o α and β tubulin strandsStabilizing or destabilizing MTs o Microtubule associated proteins (MAPs)  Bind to free ends of MTs and stabilize ends selectively to polarize a cell o Drugs can be used to change MT stability  Colchicine binds free tubulin to prevent polymerization; MTs disintegrate and mitosis stops  Taxol prevents loss of subunits from MTs; MTs become “frozen” in place and mitosis stops.MT organized transport o Anterograde transport, retrograde transport.Motor proteins use ATP to power transport along the MT railroad o Kinesin and dynein are dimers that walk along microtubule. o One ATP is used per step.Cilia and flagella are made of MTs Actin Filaments:Control cell movementFound in: o Epithelial cell microvilli o Stress fibers in cultured cells o Leading edge lamellipodia o Contractile ring in dividing cells – cytokinesisActin polymerization requires ATP o Free G-actin monomers use ATP to become F-actin to form filaments. To uncoil, hydrolyze ATP and fall apart.Actin dynamics provide force for membrane movement o ARP complex create branches
  15. 15. o Depolymerizing protein promotes ATP hydrolysis o Capping proteins cap the ends and stabilize ATP bound monomer, stabilizing leading edge.Actin binding proteins link actin fibers to the membrane and othercellular componentsIntegrins link actin to focal adhesions o Binds to extracellular structures, messages to actin.Cells move by actin crawling (dynamics)Axon growth cone crawlingRho family GTPases control actin dynamics o RhoA causes stress fibers  Stabilize actin filaments  Induces myosin phosphorylation and thus contractility o Cdc432 causes filopodia extension  Promotes actin nucleating by ARP complexes o Rac promotes lamellipodia extension  Promotes actin nucleation, but also uncapping to allow more sites of nucleation o Cell surface receptors modulate Rho family activity  Attractive cues activate Rac and Cdc42 on area of growth cone  Repulsive cues activate RhoA  Growth cone turnsMyosins: actin motor proteins o Head, neck, tail o Tails link up together o Work as dimers o Moves membranes or cell componentsMuscle contraction by actin and myosin o Myosin heads climb up actin filament o Z disks move together, muscle contracts
  16. 16. Lecture 40:Interphase G1 phase o Rest phase o Indeterminate length o Cells that are not growing go to G0 S phase o DNA replication phase G2 phase o Relatively short o Cells take a breather between replicating DNA and getting ready to enter mitosis Mitosis Nuclear division, cytokinesis Prophase: o Mitotic spindles form Prometaphase: o Chromatids start to line up on microtubules that form between two centrosomes o Break down of nuclear envelope (lamins intermediate filament) Metaphase: o Chromosomes aligned along midway of spindle o Kinetochores of all chromosomes get aligned Anaphase: o Microtubules pull sister chromatids apart o Spindle poles get shorter Telophase: o Nuclear envelope starts to divide/form o Contractile ring made of actin and myosin Cytoskeletal changes: Nuclear envelope breakdown o Phosphorylation of lamins proteins causes them to lose affinity for each other, and envelope starts to break apart. MTs form mitotic spindle in prophase
  17. 17. o Centrosomes duplicated during interphase separate and nucleate more MTs. MT instability increases because MAP activity decreases o MTs from both poles grow to meet o 3 classes of MT make mitotic spindle  Astral microtubules – not attached to anything  Kinetochore microtubules – in middle, attach to kinetochores  Interpolar microtubules – push two sides of cell apart in telophase. Where they meet in middle, joined together by motor proteins (kinesin and dynein)Movement in anaphase o Kinesins on interpolar MTs push poles apart and pull chromatids across poles o Dyneins on astral MTs pull poles toward membranesContractile ring enables cytokinesis o Ring forms of overlapping actin and myosin filaments o Ring contracts to pinch off membrane The cell cycle is controlled by cyclins and cyclin-dependent kinasesCdks phosphorylate cell targets that drive entry to different parts of cellcycleCdk activity requires cyclin binding to form Cdk complexesCyclins “cycle” through different concentrations depending on whenthey are needed4 types of cyclines, D, E, A, B o D = G1 phase o E = G1/S o A = S phase o B = G2 phaseRegulation of Cdk Complexes o Cyclin concentration  Cyclin protein expression  Degradation of existing cyclin o Cdk phosphorylation controls activity
  18. 18. Activating and inactivating kinases and phosphatases act on Cdk to regulate activity o Cdk inhibitor proteins can inhibit Cdk-cyclin complex formation o Check points:  G2M checkpoint to enter mitosis  Checkpoint between anaphase and cytokinesis by anaphase promoting complex  G1S checkpoint to start replication -> called start checkpoint. “master checkpoint” Mitogens control entry into S phase and mitosis o Receptors bind to Ras, which activates MAPK cascake to activate MAP kinase. o Goes to nucleus and phosphorylates transcription factors that activate immediate early gene expression. o IEG expression upregulates transcription of delayed genes Main role of G1-CDK is to activate E2F o E2F = transcription factor that drives transcription of other genes for S phase. o Retinoblastoma holds E2F inactive until phosphorylated by G1-Cdk DNA damage halts cell cycle by activating p53 o Stops entry into S phase and ultimately mitosis Replicative cell senescence o Telomerase replaces the ends of chromosomes (telomeres with each cycle) o Animal somatic cells have low telomerase. After a while, shortened telomeres are recognized by p53 as damaged and cell cycle is suspendedCancer cells often have increased telomerase or loss of p53
  19. 19. 41: Apoptosis (programmed cell death)Plays an important role in multicellular developmentIs it involved in deletion of entire structures, sculpting of tissues, andregulates the neuron numberCellular interactions regulate cell death in two fundamentally differentways o Most cells require signals (trophic factors) to stay alive and will undergo programmed cell death in the absence of these signals o Some cells are triggered to undergo programmed death by signalsMajor way to sculpt tissues during development (neurons, digits)Allows for normal cell turnover (epithelia, immune cells)Removes damaged cells (DNA damage)Morphological changes o Cell shrinkage o Chromatin condensation o Membrane blebbing o Nuclear fragmentation o Formation of apoptotic bodies o No cell lysisStages o A cell receives a signal that is either extrinsic or intrinsic o Cell responds to signal by activating signal transduction pathways that cause release of cytochrome c from mitochondria  Cytochrome C binds caspase complexes and causes their activation  Caspases digest cellular proteins, causing death  Caspases exist inactively as procaspaces and are activated by cleavage  Genetic loss of apoptosis proteins causes faulty developmentTriggers o Deprivation of survival factors – most cells require positive signals to stay alive.
  20. 20. o Activation of death receptors – cells have receptors that respond to extracellular ligands to signal apoptosis  FAS ligand activates FAS receptor. FADD adaptor protein binds to death effector proteins which cause complex to be set up, downstream execution of apoptosis. o Intrinsic signals – DNA damage or senescence triggers cell deathHow survival factors inhibit apoptosis o Bcl2 – inhibits cytochrome C release from mitochondria, thus inhibiting apoptosis o Can make more Bcl2, survival factors can make more Bcl2 o The point is that if you remove the survival factor, balance tips towards apoptosis. Cancer cellsProliferate without restraintIgnore signals from cell-cell and extracellular contactsResistant to apoptotic signalsCan degrade the extracellular matrix to move outside their designatedareaTypes of cancer o Carcinomas – arise from epithelial cells o Sarcomas – connective tissue or muscle cells o Leukemias or lymphomas – white blood cells o Various nervous system tumors (something-oma, ieglioma or neuroblastoma)Most common cancers are from epithelial tissues (carcinoma)Come from accumulated DNA mutations in dividing cells o Cancer is a stem cell disease from accumulated motations. Each tumor is clonal o A tumor is benign if it stays in its tissue (proliferative but still contact inhibited) o Malignant if it can break out of its niche o Metastatic if it can colonize other tissues/sites
  21. 21.  Digests through basal lamina, through capillaries, and spreads to other tissues.  Game.Over. Two types of cancer-associated genes o Proto-oncogenes  Genes whose proteins promote cell growth or motility and promote tumorogenesis when hyperactivated (iemyc, src, ras) o Tumor suppressor genes  Genes whose protein products limit cell growth or survival such that the cell is released from restraint when they are inactivated by mutation (ieRb, p53) o 7 types of proteins that participate in controlling cell growth  growth factors  growth factor receptors and intracellular receptors  intracellular transducers  transcription factors  anti apoptosis proteins  cell cycle control proteins  DNA repar proteinsCancer genes can be mutated in several ways o Point mutation o Gene amplification o Chromosomal translocation or deletionEpithelial to mesenchymal transition o Most cancers are epithelial in origin, but epithelial cells are kept in well-structured sheets o To escape, tumor cells must adopt a more mesenchymal phenotypeEMT change o Cells become less adherent, with more flexible cytoskeletons o Happens in development
  22. 22. Lecture 42: Wound HealingWhy is wound healing important to dentists?Soft tissue wound healing o After treatment for disease (periodontitis) o Post surgical healing o In response to dental materialsBone healing o After traumatic fracture o Post surgical healingGingival Wound HealingInflammationGranulation tissue formationAngiogenesisWound contraction/fibroblast migration, and remodelingRe-epithelializationClotting -> inflammation -> proliferation and migration -> functionalrestoration -> remodelingScars are when fibroblasts remain active over long periods of timesFibroblasts respond to growth factors, then respond to TGF Beta-1 andbecome differentiated myofibroblast, and now make smooth muscleactin.Cell MigrationReorganization of the actin cytoskeletonThree major types of filamentous structures o Lamellipodia o Filopodia o Actin-myosin filament bundles/stress fibersDermal RepairRemoval of damages collagen fibers by macrophagesProliferation and migration of fibroblasts into wound siteWound contractionProduction of new collagen fibersEpithelial RepairProliferation of basal keratinocytes in undamaged area around edge ofwoundScab formation (on top of clot)
  23. 23. Migration of keratinocytes under edges of scab Further proliferation recreates multiple cell layers Late stage epidermal repair of skin wound Wound penetrates through dermis to hypodermis containing adipose cells Epidermis heals under scab that is ready to detach Dermis will gradually reestablish itself Early part of restoration of functional healing has no rete pegs/dermalpapillae Large full thickness wounds Deepest part of hair follicles and sweat glands remain as islands of epithelial cells in dermis and can divide and migrate onto the surface Massive destruction of all epithelial structurs (ie, third degree burns) prevent re-epithelialiation-requires grafting or very slow epithelialization from edges of wounds Hard Tissue Bone Healing Bone remodeling induced by stress fracture/osteocyte signaling o Removal of bone lining cells; unmineralized osteoid o Fusion of monoctes into osteoclasts o Resorption of bone matrix o Recruitment of osteoblasts o New osteoid formation; mineralization o Bone Remodeling Unit Fracture Repair: Bone Cells o Bone marrow stromal cells o Periosteal cells o Hematopoietic cells o Chondroblasts o Osteoblasts o Osteoclasts Cellular Events in Bone Frcture Healing o Bleeding from damaged bone o Clot formation in space between bones (hematoma) o Coagulation cascade leading to acute inflammatory response o Proliferation of periosteal cells around hematoma
  24. 24. o Formation of cartilage at site of hematoma  Cells make cartilage ECM o New bone formation at fracture site  Appearance of new capillaries from periosteum  Endochondral ossification (woven bone)  Osteoclast resorption of woven bone and deposition of lamellar boneDealing With Bone Loss or Bone Deficiences Using Added Bone o Onlay grafting  Using bone or bone substitutes to fill bone gaps (ie from tooth extraction, or to create alveolar bone height) o Facial advancement and lafort procedures  Moving the face forward  Filling the gap with bone or bone substitutes o Distraction Osteogenesis  Lengthening bones  Widening palates o Bone transport osteogenesis  Filling bone gapsSources of bone for onlay grafting o Autologous bone  Autograft – rib, hib, fibula o Heterologous bone  Isograft – taken from identical twin  Homogfraft – from individual of same species  Allogravt – banked cadaver bone  Xenograft/Heterograft – from other speciesPrinciples of distraction osteogenesis o Use our knowledge of fracture healing to create more bone o Can be used to lengthen bone and widen palates o Stage  Attachment of distraction device, on either side of fracture, palate, or region to be distracted  If no fracture, create osteotomy  Hold region stable for 3-7 days  Begin distraction at 1-1.5mm/day
  25. 25.  After completion of distraction, hold region stable for 2X length of distraction time
  26. 26. Lecture 43: BMP Signaling, EndMTWhat is Endothelial MesenchymalTransition? What is FOP? FibrodysplasiaOssificansProgressiva o Great toe malformations o Progressive heterotopic ossification in characteristic anatomic patterns -> formation of a second skeleton o Associated with dysregulation of BMP signaling in soft tissues o Transformed into ribbons, sheets, plates of heterotopic bone through an endochondral process o Joints progressively locked in place, movement blocked o Begins in childhood, induced by trauma to tissues o Not transdifferentiation, but metamorphosis  Soft tissue destroyed, replaced with skeletal o Three forms:  Classic (toe malformations, second skeleton)  Atypical classic features plus one or more atypical (growth retardation, persistence of primary teeth)  Variant (major variations in one/both classic features – severe malformations, digit reductions, sparse nails, hair) o Genetic basis of the disorder?  G->A mutation What is the molecular lesion? o R206H mutation lies on fringe of GS regulatory subdomain o Arg206: conserved basic residue adjacent terminus of the Gly-Ser regulatory subdomain of type I receptors Linked to ALK2 and is predicted to lead to dysregulation of BMP signaling What experimental evidence supports this hypothesis? o There is none? o Gene replacement in mice to get to germ line o Heterotropic bone formation in conditional constitutively active ALK2 mouse model of FOP o Mice are stillborn if born with FOP Can heterotopic bone formation be blocked?
  27. 27. o Small molecule ATP analogs competitively inhibit ALK2 and the other BMP receptor kinasesALK2-mediated EndMT:Transition of endothelium to cartilage and boneCould formation of heterotopic bone in patients with FOP be caused byEndMT? o Cells from bony lesions and caALK2-transgenic mice express marker proteins specific for endothelium o There is an endothelial origin in bony lesions o Engeineered genes can reveal when and where a gene is expressed -> reporter construct  GFP can be used to identify specific cells in a living animalDoes the mutation in ALK2 cause EndMT? Is the mutation sufficient? o EMT prevalent in many cancers. o One point mutation is sufficient to introduce morphological change in cell lines.Are the entothelial-derived mesenchymal cells multi potent stem likecells? o The answer is a resounding yes. o ALK2 mutant forms different multipotent stem-like cells!!! Dun dundun.Can the endothelial-derived mesenchymal cells be employed forregenerative purposes in vivo? o Implanted into mice o Cells adopted anticipated fates through implantation. o Polylactic acid sponges are what is implanted. Summary:EndMT generates mesenchymal stem-like cells that can differentiateinto multiple linagesActivation of ALK2 is necessary and sufficient for EndMT to occur incells such as HUVECs and HCMECs under in vitro conditions of studyFOP, with hallmark pathological bone formation, is a vascular diseasebased on conversion of endothelial cells into mesenchymal stem likecells