Lecture 36: Membranes and Membrane Proteins11/26/11 4:25 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

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 (adenyl cyclase)

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

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.

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

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

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

cAMP phosphodiesterase

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.

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

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

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

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

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

o

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 = epidermolysis bullosa 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.

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

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

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

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

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

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.

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, ie glioma 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

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 (ie

myc, 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 (ie Rb, 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

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)

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

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

After completion of distraction, hold region stable for 2X

length of distraction time

Lecture 43: BMP Signaling, EndMTWhat is Endothelial Mesenchymal

Transition?

What is FOP?

Fibrodysplasia Ossificans Progressiva

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?

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

dun dun.

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