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. o Glycolipids found on outside
o Phosphatidyl-serine, inositol, ethanolamine found on inside
usually.
Flippases transfer phospholipids to other side of membrane
New membranes are synthesized from ER.
o Form vesicles which fuse with other membranes
Membrane Proteins
Polypeptide chain usually crosses the bilayer as an α-helix
Proteins can be solubilized in detergents and purified
Plasma membrane is reinforced by the cell cortex
Cell surface is coated with carbohydrate
Cells can restrict movement of membrane proteins
Functions
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 protein
molecules.
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 polar
backbone is exposed
Multiple alpha helixes form a hydrophilic pore
Porin proteins form water-filled channels in the outer membrane of a
bacterium
o Formed by 16 strands of β-sheets
o Allows passage of ions and nutrients across outer membranes
of some bacteria and of mitochondria
Membranes are disrupted by detergents such as SDS and Triton X-100
o Have only one tail
Bacteriorhodopsin acts as a proton pump powered by light, drives ATP
synthase

3. Plasma membrane reinforced by cell cortex – imparts shape and
function
o Spectrin meshwork forms the cell cortex in red blood cells
Eukaryotic 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 infection
Movement 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. Lecture 37
I. 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. 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 receptors
or 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 wall
Steroid hormones bind intracellular receptors that act as gene
regulatory 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 protein
Most signal molecules bind to receptor proteins on the target cell
surface
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 cells
Many 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 Receptors
Stimulation of G-protein linked receptors
G proteins can regulate ion channels

6. G proteins can activate membrane bound enzymes
Cyclic AMP pathway can activate downstream genes
Inositol phospholipid pathway triggers rise in Ca
Ca signal triggers many biological processes
Intracellular signaling cascades can achieve astonishing speed (ie
photoreceptors 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 GTP
G proteins couple receptor activation to opening of cardiomyocyte K
channels
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 intracellular
second 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
cAMPphosphodiesterase
Extracellular signals can act rapidly or slowly
Rise in intracellular cyclic AMP can activate gene transcription through
protein kinase A
o Translocates through nuclear pore, into nucleus,
phosphorylates gene regulatory protein to activate target
gene.

7. Membrane bound phospholipase C activates two small messenger
molecules: 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 -> secretion
Calcium/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 receptors
Activated receptor tyrosine kinases assemble a complex of intracellular
signaling 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 cascade
Some enzyme-linked receptors activate a fast track to the nucleus
Protein kinase networks integrate info to control complex cell behaviors
Multicellularity and cell communication evolved independently in plants
and animals

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 at
the plasma membrane
Signaling pathways can be highly interconnected: cross-talk
Lecture 38
General introduction:
Membrane enclosed organelles are distributed throughout the
cytoplasm
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 through
invagination of plasma membrane.
Mitochondria are thought to have originated from aerobic prokaryote
being engulfed by a larger anaerobic eukaryotic cell -> has it’s own
genome.
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 mRNA
One Endoplasmic Reticulum

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 cells
Golgi 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 off
Other 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
molecules
Protein 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.  By vesicles: from ER onward and from one
endomembrane compartment to another ferried by
transport vesicles
Protein 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 organelles
ER: 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 embedded
Vesicular 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 communication
Protein transport: quality control
o Most proteins that enter ER are destined for other locations

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 retained
Exocytosis
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 signal
Endocytosis
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. o

13. Lecture 39: Cytoskeleton
Roles 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. Organize cell organelles and control traffic of vesicles
Roles 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 strands
Stabilizing 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 movement
Found in:
o Epithelial cell microvilli
o Stress fibers in cultured cells
o Leading edge lamellipodia
o Contractile ring in dividing cells – cytokinesis
Actin 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. 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 other
cellular components
Integrins link actin to focal adhesions
o Binds to extracellular structures, messages to actin.
Cells move by actin crawling (dynamics)
Axon growth cone crawling
Rho 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 turns
Myosins: actin motor proteins
o Head, neck, tail
o Tails link up together
o Work as dimers
o Moves membranes or cell components
Muscle contraction by actin and myosin
o Myosin heads climb up actin filament
o Z disks move together, muscle contracts

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. 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 membranes
Contractile 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 kinases
Cdks phosphorylate cell targets that drive entry to different parts of cell
cycle
Cdk activity requires cyclin binding to form Cdk complexes
Cyclins “cycle” through different concentrations depending on when
they are needed
4 types of cyclines, D, E, A, B
o D = G1 phase
o E = G1/S
o A = S phase
o B = G2 phase
Regulation of Cdk Complexes
o Cyclin concentration
 Cyclin protein expression
 Degradation of existing cyclin
o Cdk phosphorylation controls activity

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 suspended
Cancer cells often have increased telomerase or loss of p53

19. 41: Apoptosis (programmed cell death)
Plays an important role in multicellular development
Is it involved in deletion of entire structures, sculpting of tissues, and
regulates the neuron number
Cellular interactions regulate cell death in two fundamentally different
ways
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
signals
Major 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 lysis
Stages
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
development
Triggers
o Deprivation of survival factors – most cells require positive
signals to stay alive.

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
death
How 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 cells
Proliferate without restraint
Ignore signals from cell-cell and extracellular contacts
Resistant to apoptotic signals
Can degrade the extracellular matrix to move outside their designated
area
Types 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.  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 proteins
Cancer genes can be mutated in several ways
o Point mutation
o Gene amplification
o Chromosomal translocation or deletion
Epithelial 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
phenotype
EMT change
o Cells become less adherent, with more flexible cytoskeletons
o Happens in development

22. Lecture 42: Wound Healing
Why 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 materials
Bone healing
o After traumatic fracture
o Post surgical healing
Gingival Wound Healing
Inflammation
Granulation tissue formation
Angiogenesis
Wound contraction/fibroblast migration, and remodeling
Re-epithelialization
Clotting -> inflammation -> proliferation and migration -> functional
restoration -> remodeling
Scars are when fibroblasts remain active over long periods of times
Fibroblasts respond to growth factors, then respond to TGF Beta-1 and
become differentiated myofibroblast, and now make smooth muscle
actin.
Cell Migration
Reorganization of the actin cytoskeleton
Three major types of filamentous structures
o Lamellipodia
o Filopodia
o Actin-myosin filament bundles/stress fibers
Dermal Repair
Removal of damages collagen fibers by macrophages
Proliferation and migration of fibroblasts into wound site
Wound contraction
Production of new collagen fibers
Epithelial Repair
Proliferation of basal keratinocytes in undamaged area around edge of
wound
Scab formation (on top of clot)

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/dermal
papillae
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. 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 bone
Dealing 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 gaps
Sources 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 species
Principles 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.  After completion of distraction, hold region stable for 2X
length of distraction time

26. Lecture 43: BMP Signaling, EndMTWhat is Endothelial Mesenchymal
Transition?
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. o Small molecule ATP analogs competitively inhibit ALK2 and
the other BMP receptor kinases
ALK2-mediated EndMT:
Transition of endothelium to cartilage and bone
Could formation of heterotopic bone in patients with FOP be caused by
EndMT?
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
animal
Does 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 like
cells?
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 for
regenerative 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 differentiate
into multiple linages
Activation of ALK2 is necessary and sufficient for EndMT to occur in
cells such as HUVECs and HCMECs under in vitro conditions of study
FOP, with hallmark pathological bone formation, is a vascular disease
based on conversion of endothelial cells into mesenchymal stem like
cells