endocytosis

1
Exocytosis and endocytosisAnne Kenworthy,
Ph.D.Dept. of Molecular Physiology and
Biophysics718 Light Hall
Research Interests
GFP-CVLS
GFP-NRas

• Trafficking of Ras in the secretory and endocytic
  pathways
• Organization and dynamics of membrane
  microdomains and their role in Ras signaling
• Live cell imaging
• Microscopy-based methods to measure protein
  dynamics and protein-protein interactions in
  living cells

2
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

3
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

4
Getting molecules into cells crossing the plasma
membrane

• Diffusion across the plasma membrane
• water, gases, small molecules
• Protein-mediated transport
• ion channels, transporters
• Pore formation
• toxins
• Membrane fusion
• viruses
• Formation and internalization of membrane-limited
  vesicles
• Endocytosis
• Pinocytosis
• Phagocytosis

extracellular
??2?
O2
Y
Y
O2
??2?
Y
cytoplasm
5
Endocytosis

• Small region of the plasma membrane invaginates
  to form membrane-limited vesicles (0.05-0.1 mm
  diameter)
• Internalized molecules retain topology (lumenal
  extracellular)
• Cargo can be specifically selected
• Destination of cargo can be controlled
  destination depends on pathway of uptake
• Cargo receptors can be recycled to the cell
  surface
• Conditions under which internalization occurs can
  be regulated

6
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

7
Non-clathrin Non-caveolae
Clathrin-mediated
Caveolae
Macropinocytosis
caveolae
CCV
EE early endosome RE recycling endosome CCV
clathrin-coated vesicle PMplasma membrane
Non-CCV
RE
Golgi
Macro-pinosome
EE
caveosome
lysosome
late endosome
ER
nuclear envelope
Sieczkarski and Whittaker 2002 J. Gen. Virol.
831535
8
1. Clathrin-mediated endocytosis
17-44
9
Compartments and pathways in clathrin-mediated
endocytosis
10
2. Caveolae
11
Caveolar endocytosis
Early endosome
Intermediates (caveosomes)
Recycling endosome
Nichols and Lippincott-Schwartz (2002) Trends
Cell Biol. 11406
12
Caveolar endocytosis

• Caveolae are 50-100 nm invaginations on the cells
  surface
• Caveolin, a membrane protein, is the coat
  protein of caveolae
• Do not undergo constitutive endocytosis but can
  undergo endocytosis in response to a signal (ex.
  SV40 binding) in a cholesterol- and
  dynamin-dependent fashion
• Internalized caveolae recruit actin to form
  comet tails
• Upon internalization caveolae are delivered to
  novel endosomal compartments known as
  caveosomes

13
3. Non-clathrin/non-caveolae

• Currently poorly understood
• Defined by process of elimination
• Multiple mechanisms for testing for
  clathrin-mediated uptake mechanisms
• Both caveolae and clathrin-mediated endocytosis
  involves dynamin
• Caveolae endocytosis can be inhibited by dominant
  negative caveolin
• Caveolae endocytosis is inhibited by cholesterol
  depletion
• Emerging theme involves detergent-resistant
  membranes/ lipid rafts

14
Lipid rafts

• Membrane microdomains enriched in glycolipids and
  cholesterol
• Function by segregation some proteins are
  enriched in rafts, others are excluded
• Participate in TGN-to-PM trafficking of
  apically-destined proteins
• Function at the PM in endocytosis and in
  organizing cell signaling pathways
• Controversial model

Simons and Ikonen (1997) Nature 387569
15
4. Pinocytosis/ phagocytosis

• Pinocytosis internalization of fluid
• Actin dependent
• Generated at sites of ruffling at the plasma
  membrane
• Includes macropinocytosis (vesicles gt 1 mm in
  diameter) and micropinocytosis (vesicles lt 200nm
  in diameter)
• Phagocytosis internalization of particles
• Occurs in specialized cells ex. neutrophils and
  macrophages
• Actin dependent
• Clathrin independent
• Particles gt 0.5 mm in diameter

16
Phagocytosis vs. clathrin-mediated endocytosis
5-44
17
How can you tell if endocytosis is clathrin
mediated? Should be inhibited by

• Overexpression of mutant forms of coat proteins-
  dominant negatives (DN) interfere with function
  of normal proteins
• DN Dynamin K44A (also inhibits caveolae)
• DN Eps15- binds AP-2, arrests coat assembly
• Clathrin hub domain overexpression
• Expression of m2 subunit of AP-2 (affects YXXØ
  but not di-leucine containing proteins)
• Potassium depletion or incubation in hypertonic
  medium- interfere with clathrin coat assembly
• Typically use a marker for this pathway (ex.
  transferrin) as a positive control in conjunction
  with these treatments to show they are effective

18
How can various endocytic routes be further
differentiated experimentally?

• clathrin-dependent
• caveolar
• lipid rafts/dynamin dependent
• lipid rafts/ dynamin independent
• Non-clathrin/non-lipid rafts

Pelkmans and Helenius (2003)
19
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

20
Some of the functions of endocytosis

• Nutrient uptake
• Receptor recycling
• Plasma membrane protein downregulation and/or
  degradation
• Synaptic vesicle recycling
• Transcellular signaling
• Exploitation virus and toxin entry into cells

21
Endocytosis of LDL receptors

• Low density lipoprotein (LDL) particles are a
  carrier for cholesterol
• LDL particles are taken up by clathrin-mediated
  endocytosis
• Mutant LDL receptors in patients with familial
  hypercholesterolemia are defective in
  internalization
• ex. tyrosine to cysteine change in YXXØ motif in
  cytosolic domain
• The ligand and receptor separate in late
  endosomes due to low pH
• The LDL receptor is recycled to the cell surface
• The LDL particle is broken down in lysosomes

17-46
22
Endocytosis of transferrin receptors

• Transferrin is a glycoprotein that binds and
  transports iron
• Apotransferrin iron-free
• Ferrotransferrin two bound iron atoms
• Transferrin receptors bind ferrotransferrin at
  neutral pH
• At low pH (of late endosomes), iron releases from
  transferrin while apotransferrin remains bound to
  the receptor
• Apotransferrin and the transferrin receptor are
  recycled to the cell surface where apotransferrin
  is released

17-48
23
Removal of activated receptors from the cell
surface i.e. receptor downregulation

• Ligand binding induces receptor internalization
• Targets receptors for degradation
• Some receptors can continue to signal until they
  are incorporated into multivesicular bodies

Sorkin and Zastrow (2002) Nature Reviews
Molecular Cell Biol. 3600
24
Some viruses like influenza exploit the low pH in
endosomes as a fusion trigger
25
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

26
Key steps in the formation of clathrin-coated
vesicles
Activation (TGN)
Activation (PM)
Cargo capture
Arf-1
AP-2
AP-1
SNARE
ReceptorsYXXf/LL
arrestin
Docking complex(?)
Hsc70
Coat assembly
Scission
Uncoating
clathrin
Auxilin
dynamin
dynamin
amphiphysin
Kirchhausen 2000 Nature Reviews Molecular Cell
Biology 1187
27
Making and moving vesicles general sorting and
trafficking machinery

• Cargo sorting signals
• Membrane lipids
• Vesicle formation- clathrin and accessory
  proteins
• Cargo capture- adaptors
• Pinchase- dynamin
• Direct vesicle movement- actin, microtubules and
  motors
• Vesicle targeting and fusion machinery- Rabs,
  SNARES

Marsh and McMahon (1999)
28
Sorting signal for clathrin-mediated endocytosis

• AP-2 is the PM clathrin adaptor
• Sorting signals for incorporation into clathrin
  coated pits include di-leucine motifs and YXXØ
• Many other regulatory proteins involved in
  clathrin-mediated endocytosis
• Sorting signals into non-clathrin endocytic
  pathways are not well understood

29
Protein-protein interactions that suggest a link
between endocytic and cytoskeletal machinery

• Actin filaments faciliate clathrin-mediated
  endocytosis under some conditions but disruption
  of filaments does not universally inhibit vesicle
  formation in higher eukaryotes
• Genetic evidence in yeast indicates a link
  between actin and endocytosis
• Actin tails associate with some endocytic
  vesicles

Schafer (2002) Curr. Opin. Cell Biol. 1476-81
30
Endocytosis

• Why do cells need endocytosis?
• Is there more than one endocytic pathway ?
• Clathrin-mediated uptake
• Caveolae
• Non-clathrin/non-caveolae pathways
• Pinocytosis/ Phagocytosis
• What are the functional consequences of
  endocytosis?
• How are endocytic structures formed and how do
  they know where to go?
• Where do the textbook models come from?

31
Example from the literaturedefining a
clathrin-independent internalization pathway

• How does cholera toxin get inside cells?

32
Cholera toxin modifies Gsa subunits and
constitutively activates adenylyl cyclase

• Cholera toxin binds to and enters intestinal
  cells
• Once inside the cell, it covalently modifies a
  heterotrimeric G-protein
• Leads to persistant adenylyl cyclase activation,
  elevation of cAMP, and chloride secretion
• Causes diarrhea

33
How does cholera toxin (CTX) get to the right
place to do its dirty work?

• Cholera toxin binds a glycolipid, GM1, on the
  cell surface
• AB5 structure
• B subunit (CTXB) membrane binding
• A subunit catalytic
• Cholera toxin must somehow cross the plasma
  membrane to interact with Ga subunits (which are
  located on the other side of the membrane!)

Cholera toxin
20-16
34
Endocytosis and retrograde transport of bacterial
protein toxins
Cholera toxin A subunit contains a KDEL sequence
that allows it to be transported from the Golgi
complex to the ER. Once it reaches the ER, it
crosses the translocon to enter the cytosol. But,
how exactly does it get the the Golgi?
Falnes and Sandvig (2000) Curr. Opin. Cell Biol.
12407
35
Is cholera toxin internalized to the Golgi
complex by a clathrin-dependent process?

• Epsin and eps15 mutants inhibit clathrin-mediated
  transferrin (Tf) uptake to recycling endosomes
• Epsin and eps15 mutants do not affect cholera
  toxin B-subunit (CTXB) uptake to the Golgi
  complex (marked by b-COP)
• Suggests CTXB is delivered to the Golgi complex
  by a clathrin-independent pathway

b-COP Marker for the Golgi complex
36
Does internalized CTXB pass through early
endosomes?

• Early endosome function requires the GTPase Rab5
• Dominant negative rab5 S34N (GDP bound)
  expression perturbs early endosomes and blocks
  transferrin uptake
• Rab5 S34N does not affect delivery of CTXB to the
  Golgi complex
• Suggests CTXB does not pass through early
  endosomes

Nichols et al. 2001 J. Cell Biol.
37
Current questions

• What are the physiological cargo carried by
  non-clathrin mediated endocytic pathways?
• How does cholera toxin get targeted into
  caveolae/ lipid rafts?
• What role does caveolin play in the
  internalization of cholera toxin?
• Do clathrin-mediated and non-clathrin mediated
  endocytic pathways intersect?
• What cellular machinery is responsible for
  non-clathrin pathways?