Post on 22-Dec-2015
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