Announcements

Review sessions here Friday 11:50-1:10, Monday 6-8PM

Exam next Wednesday covers through endocytosis today

Protein targeting vs vesicle targetingProtein targeting = loading protein into correct vesicle type

Soluble proteins bind receptor which is recruited into appropriate vesicle by adaptin or COP

Examples: KDEL on ER resident proteinsmannose-6-P on lysosomal proteinsLDL binds LDL receptor

Membrane proteins (and receptors) are loaded into correct vesicle by binding to adaptin or COP

Examples:KKXX on KDEL receptor binds COPmannose-6-P receptor binds adaptinNPVY on LDL receptor binds adaptin

Vesicle targeting to correct organellev and t SNAREs and Rabs and ???

Recruitment of LDL-R to coated pits requires an “endocytosis signal” in cytoplasmic domain

Adaptin complex (four polypeptides)

Plasma membrane

Adaptin complex binds endocytosis signal in cytoplasmic domain of receptor:

-NPXY- (Asn-Pro-Val-Tyr) in LDL-R

Adaptins recruit clathrin and initiate coated pit/vesicle formation

Val

Tyr

Pro

Asn

LDL-R

Endocytosis signal

LDL

Based on MBoC (3) figure 13-53

HOOC

familial hypercholesterolemia–Mutations in N-terminal domain:

LDL-R doesn’t bind LDL–Mutations in C-terminal domain:

LDL-R is not internalized

Summary of “receptor-mediated” endocytosis of LDL

ATP

ADP+Pi

H+

Lysosome

Early endosome

ATP ADP+Pi

Uncoating(HSP70 family)

GTP

GDP+Pi

Coatedvesicle

Fusion(Snares)

Cholesterol released for membrane synthesis

A single receptor makes hundreds of trips (~10 min/cycle)

Free cholesterol

pH ~7.2pH ~6

LDL-R

pH ~7-.7.2Low density lipoprotein (LDL)

Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptor

dynamin

15.12-receptor_endocytosis.mov

A single coated pit has many different receptors and cargos

1,000s of receptors of many types per coated pit…

Same coated pits used for pinocytosis!

LDL-R

Low density lipoprotein (LDL)

Coats for all reasons: a summary of vesicle coats and functions

COPs:COPII: ER to Golgi transport, intra-Golgi (Sar1 GTPase)COPI: intra-Golgi, Golgi to ER, Golgi to plasma membrane, early to late/lysosome

Clathrin:Plasma membrane to early endosome (endocytosis)Golgi to endosome/lysosome

Endosomes sort internalized receptors and ligands

Transcytosis - movement of receptor to a different membrane from the one in which it was endocytosed

Mainly receptors are recycled (LDL receptor)

Mainly ligands are degraded (LDL)

Maternal IgG–Secreted IgA–Others

ECB 15-33

“Transcytosis” moves maternal IgG across epitheliaIntestinal lumen

Apical membrane

Endosome

Endosome

Basolateral membrane

Milk duct

Maternal bloodNeonate blood

IgG is transported across the mammary epithelium into milk by transcytosis

Receptor-mediated endocytosis from basolateral domain

Secretion from apical membrane domain

Epithelial cell

IgG in milk

IgG receptor

IgG receptor

Basolateral

Apical

IgG in blood

IgG is transcytosed into the neonate blood Endocytosis from apical domain and secretion to basolateral membrane

Polarized epithelial cells have distinct apical and basolateral endosome compartments

Membrane flow during exocytosis and endocytosis is a delicate

balance

Endocytosis internalizes membrane ~2-3% per minute…

Entire membrane is recycled in less than 1 hr…

Block endocytosis, exocytosis continues:

Block exocytosis, endocytosis continues:

Lysosome

ER

Golgi apparatus

Endosome

membrane area shrinks…

membrane area grows…

Original surface

Protein targeting and trafficking, finale!

Cytoplasm

Secretion/membrane proteins

Secretory vesicles

Lysosomes

Endosomes

RetrievalTransport

(constituitive secretion)

(regulated secretion)

Pro

tein

ta

rgeti

ng

Vesi

cle t

arg

eti

ng

RER

Golgi

Plasma membrane

Signal sequence

KDEL (soluble proteins)

KKXX (membrane proteins)

M6P

Nucleus NLS: (basic)

NES: (L-rich)

Mitochondria

Chloroplasts

Default

Signal peptide

Signal peptide

Endocytosis: From plasma membrane to endosome to lysosome…

PeroxisomesSKL at C term.

Endocytosissignal

Default

Microfilaments: MuscleOrganelle transport in plantsIn all eukaryotes

Microtubules:Cilia and flagellaOrganelle transport in animalsIn all eukaryotes

Intermediate filaments:Cell structure Mainly in vertebrates

“Cytoskeleton”

ECB 1-20

Next lecture…

Lecture 18: Many eukaryotic cells contain a cytoskeleton composed of three filament systems

MicrotubulesIntermediate filaments Microfilaments

25 nm 25 nm25 nm

ECB figure 17-2

25 nm dia.

Tubulins (MAPs and motors)

Vesicle transport, cell polarity, and division

9-12 nm dia.

Family of related proteins, cell type specific

Elasticity and tensile strength…(not in plants, why?)

6-7 nm dia.

Actin (myosin, and accessory proteins)

Contraction, vesicle transport, locomotion, and division

Lecture 18-20

L18-Begin with intermediate filamentsActin; muscle

Next lecture (19); non-muscle actin

Lecture 20 - Microtubules and flagella

Some examples of IFs

MTs

NFs

MCB figure 19-55 MBoC figure 16-16B

See ECB figure 17-3

Keratin filaments in epithelial cell Neurofilaments

Neurofilaments Nuclear lamina

Inside face of inner nuclear membrane

Characteristics of IFs Filament diameter is ‘intermediate’ between actin and

myosin Strong ropelike structures, gives shape to nucleus of

most eukaryotes and cytoplasm of vertebrate cells.

Provide tensile strength to resist mechanical stress

ECB 17-5

Anti-parallelstaggered tetramer

“Coiled-coil” dimer

IFs are polymers of coiled-coil subunits

8 tetramers of final filament

Assembly and dynamics often regulated by phosphorylation

ECB 17-4

NH2COOH

NH2 COOHNH2

COOH

NH2 COOH

NH2 COOH

NH2 COOH

NH2COOH

-helical monomer

Two tetramers

Intermediate filaments are a family of related proteins

all nucleated cells (except yeast?)

Sequence comparison suggests that IF proteins diverged from common ancestral protein (~35% aa identity overall, as high as ~70% in sub-

families)

ECB 17-6

Skin, hair, nails, claws

ALS(Lou Gherig’s disease)Abnormal accumulation of neurofilament

Human disorders of IFs - cells exposed to mechanical stress: epidermal keratins and EB

ECB figure 19-34 © Garland Publishing

Basal cells express “undifferentiated” keratins K5 and K14

Point mutations in K5 and K14 disrupt keratin filaments

…and cause “epidermolysis bullosa simplex.” Basal cells lyse, leading to blistering after mild mechanical trauma

Basal cells

ECB 21-37

Epidermolysis Bullosa Simplex

Lecture 18

Begin with intermediate filaments

Move to microfilaments (F-actin); muscle

Next lecture (19); non-muscle actin

Lecture 20 - Microtubules and flagella

Lecture 18: Most eukaryotic cells contain a cytoskeleton composed of three filament systems

MicrotubulesIntermediate filaments Microfilaments

25 nm 25 nm25 nm

See ECB figure 16-2

25 nm dia.

Tubulins (MAPs and motors)…

Vesicle transport, cell polarity, and division…

9-12 nm dia.

Family of related proteins, cell type specific

Elasticity and tensile strength…( mainly vertebrates)

6-7 nm dia.

Actin (myosin, and accessory proteins)

Contraction, vesicle transport, locomotion, and division

Actin and Myosin: the structure and function of muscle

“Smooth muscle” (gastrointestinal tract, bladder, uterus) is optimized for slow, steady contraction

“Myoepithelial cells” are associated with some secretory glands (mammary, sweat glands, etc)

Cardiac and skeletal muscle are referred to as “striated” muscle. Cross striations apparent by light and electron microscopy

Striated muscle is an evolutionally ancient tissue type

Cardiac muscle(cardiac myocyte) Smooth muscle cell Myoepithelial cell

Skeletal muscle (muscle fiber)

Skeletal muscle

The sarcomere is the unit of contraction in myofibril

ECB 17-42

Each myofibril contains many sarcomeres which causes striated appearance

Muscle cell is formed by fusion of precursor cells: multinucleate and contains many myofibrils (contractile

elements)

Sarcomere nomenclature

ECB 17-43 Sarcomere is Z line (Z disk) to Z line

M-line

I band A bandZone where thick and thin filaments overlap

H2N

COOH

Actin (42 kDa)

MBoC (4) figure 16-7 © Garland Publishing

Thin filaments contain actin and associated proteins

“F-(filamentous) actin”

Tropomyosin

Troponins

ATP

“G- (globular) actin” (42 kDa)Salt/Mg2+

Two other components of thin filaments{

(affects assembly dynamics)

37 nm

Two-stranded helix repeating every 13 units (37nm)

ECB17-30

Thick filaments are composed of myosin-II

150 nm

See ECB 17-40

N-terminal motor

domains of heavy chains

Light chains

C-terminus of heavy chains

Coiled-coil tail

Myosin-II (~500 kDa): 2 x 205 kDa: myosin heavy chains (MHC)

2 each of 16 and 20 kDa myosin light chains (MLCs)

Thick filaments

Filament formation via lateral aggregation of coiled-coil tails of myosin heavy chain

Heads project laterally

Thick filament is “bipolar” - myosin-IIs point in opposite directions on either side of bare zone

Bipolar

Bare zone Myosin-II heads

15 nm x 1.5 mm

MBoC figure 16-87

Actin filaments are “polarized:” “S1-decoration”

F-actin plus S1 fragments

-ATP“S1-

decoration”

“Pointed” or “minus-

end”

Slow growing

end

“Barbed” or “plus-end”

Fast growing end

Coiled-coil tail

C-terminus of heavy chains

N-terminal motor domains

Papain

“S1 fragments”

Myosin-II

Myosin is a molecular motor using ATP to “walk” along actin filaments

1. Bind myosin (or fragments thereof) to glass slide

2. Add fluorescently-labeled actin filaments

3. Add ATP

Filaments slide!

t = 0 1 2 3

Slidingactin.mov

5.

ADP

1.Myosin is an “actin-dependent” ATPase that acts as a “molecular motor”

1. No nucleotide. Myosin head is tightly bound to actin (“rigor”)2.

ATP

3.

4.

Pi

Myosin headActin filament

Thick filament

“-” “+”

“-” “+”

2. ATP binding releases myosin from actin

3. ATP hydrolysis “cocks” myosin

4. Pi is released, strengthening binding of myosin to actin

5. Myosin binds actin tightly and undergoes “power stroke” releasing ADP…

Myosin heads “walk” towards “barbed” (“plus”) -end of actin filament…

17.7-myosin.mov

Muscle contraction involves actin-myosin II sliding

ECB 17-41

Thick filaments are bipolar; myosin heads on two sides ratchet in opposite directionsBoth sides ratchet toward + end of actin (myosin is a + end directed motor)Causes actin filaments to slide in opposite directionsActin filaments don’t slide back because other myosins are bound

Contracted myofibril

Myofibril contraction results from sliding of thin and thick filaments

Thick filaments

Thin filaments

Filaments slide as myosin heads walk toward plus-ends of thin filaments (towards Z-lines)…

ZMZRelaxed myofibril

+ATP+Ca2+

Sarcomere shortens…I-bands shorten…A-bands unchanged…

I-bandA-band

A-band I-band

- +-+

Z Z

Z ZM

ECB 17-44

Filament sliding leads to contraction

Contraction is regulated by toponin and tropomyosin

Tropomyosin filament binds along actin filament

Troponin complex binds to tropomyosinIn the absence of Ca2+, tropomyosin blocks myosin binding siteIn presence of Ca2+, Troponin C binds Ca2+

Conformational change of troponins and tropomyosin uncovers myosin binding site

Myosin walks on actin and myofibril contractsRemoval of Ca2+ restores inhibition

ECB 17-48

Where does Ca2+ come from?

Myofibril contraction is stimulated by release of Ca2+ from the “sarcoplasmic

reticulum

Myofibril

Plasma membrane

T-systemT-tubules formed from invaginations of plasma membrane. The T-system carries “nerve impulse” into muscle fiber…

“Sarcoplasmic reticulum (SR)”

Derivative of ER

SR serves as a Ca2+ reservoir

Signal from neuron causes Ca2+ release thru voltage-gated Ca2+ channels.

Ca2+ stimulates myofibril contraction.

Contraction is terminated by pumping Ca2+ back into the SR…

ECB 17- 46

17.13-muscle_contraction.mov

Calcium release occurs through a voltage-gated channel

ECB 17-47