Lecture 4, BICD110
ENDOCYTOSIS
Reading, Lodish
(7th edition) =
pp 654-660, 429, bottom 647-648 (dynamin),
& health paragraphs on pp 468 & 764
(6th edition) =
pp 606-612, 393, 1023 (dynamin),
& health paragraphs on pp 709 & 432
My office hours this week are today, THURSDAY
4:00-5:30 pm in 2130 Pacific Hall
ENDOCYTOSIS
- Intro - What & Why
- Focus on Receptor-Mediated Endocytosis:
- -- Steps 1-10
-
Classical Receptor - Step 1
Clathrin
Adaptins - Steps 1-3
Pinching off - Dynamin
Uncoating
Endosomes & Acidification
Discovery of the LDL receptor & human mutations
- Fates of Ligands
“ENDOCYTOSIS”
By all eukaryotic cells
Why?
Examples:
-- All cells: for nutrition:
-Cholesterol
-Fe (iron)
All cells: for downregulation of growth factor receptor
Some cells: engulf foreign invaders
PHAGOCYTOSIS = “cell eating”
By some cells:
--> engulf foreign invaders
--> destroy them
What cells?
Macrophages --> do phagocytosis
-take up 25% of their volume/hour
-take up 3% of their plasma membrane/min
--> 100% of their PM every 30’
1) eat bacteria
2) eat dying or dead cells
--> eats 1011 old red blood cells/day
PHAGOCYTOSIS
A neutrophil phagocytosing a dividing bacterium
A mouse macrophage phagocytosing two old red blood cells
PHAGOCYTOSIS
PINOCYTOSIS = “cell drinking”
g
no concentration of contents
TYPES OF
RECEPTOR-MEDIATED ENDOCYTOSIS
**1) Clathrin -- the major type we will study
can concentrate molecules taken up 100x
2) Calveolae -- “little cavities” - calveolin
becomes a “coated vesicle”
“early”
early
Endosome
pH6
A TYPICAL RECEPTOR FOR ENDOCYTOSIS
DISCOVERY OF CLATHRIN
CLATHRIN-MEDIATED ENDOCYTOSIS
CLATHRIN-COATED PITS
THE DISCOVERY OF DYNAMIN
A Strange Fly Mutant
A temperature-sensitive (ts) mutant of Drosophila, called shibire
(= numb), that becomes paralyzed when put at high temperature.
1) shibire fly
paralyzed
raise temp
to 30oC
flies around again
lower temp
to 20oC
Shibire Mutant
Experiment:
2) Do electron microscopy on 30oC shibire fly tissues:
- Nerve terminals:
NO synaptic vesicles
- Other cells:
NO coated vesicles,
BUT lots of coated pits
& excess plasma membrane
Shibire gene --> dynamin = the pinchase
= a 100kd GTPase
- when mutant (shibirets), can’t cleave GTP &
dynamin can’t pinch to form coated vesicle
Synaptic terminal plasma membrane of a shibire fly at high
temperature. You can see coated pits trying to form into coated
vesicles, but blocked because mutant dynamin can’t cause pinching
off. Note the lines across the necks = dynamin collars.
Immuno-EM with a
gold-labeled anti-dynamin
antibody
(Adaptin =)
“early”
(= AP complex)
Identifying the “Uncoating ATPase”
Dr. Sandy Schmid
+
THE LDL RECEPTOR
Mike Brown, Joe Goldstein
Familial hypercholesteremia
hypercholesteremia
--> atherosclerosis --> heart attacks
LDL RECEPTOR MUTATIONS
-/- in their teens
-/+ in their 40’s-60’s
- that is, before statins were invented
LDL
Particle
LDL
Particles
LDL
Particle
LDL
Particle
LDL Receptor Details Revealed
- Ligand binding domain/arm
- Beta- propeller - needed for
conformational change at acid pH
- O-linked sugars - not needed
N
P
X
Y
- Transmembrane domain
- Sorting signal binds to adaptins
in coated pits
NORMALLY
AT ACID PH:
Low pH
ENDOCYTOSIS
And STORAGE
OF YOLK
PARTICLES
TRANSCYTOSIS
CALVEOLAE