Transport into the cell from
the plasma membrane:
endocytosis
The ‘road map’ of protein traffic in secretory
and endocytic pathways!
Figure 13-3a Molecular Biology of the Cell (© Garland Science 2008)
endocytosis vs. exocytosis!
Lecture: Endocytosis
1.  The plasma membrane
1.1. Functions
1.2. Domains
1.3. Layers
2.  Endocytosis
2.1. Types and definition
2.2. Endocytic vesicles; Bulk versus receptor-mediated uptake
2.3. Phagocytosis
2.4. Pinocytosis
""
"2.4.1. Clathrin-mediated endocytosis – cargo recognition
""
"
"2.4.1.1. Cholesterol uptake by LDL receptor
""
"
"2.4.1.2. Iron uptake by transferrin receptor
""
"2.4.2 Caveolae
3.  The endosomal compartment
" 3.1. Early endosomes – a molecular sorting station
" 3.2. Maturation of early to late endosomes; MVBs
Plasma membrane
•  Defines the outer boundary of the cell
•  Mediates influx and efflux of substances and information
–  Maintains intracellular ionic environment
–  Supports a positive outside membrane potential that is essential for
example for nerve conductance
–  Exchanges gases (O2 and CO2)
–  Adsorbs and releases nutrients, waste products, vitamins.
–  Internalizes fluid, macromolecules, and particles by endocytosis
–  Mediates secretion of substances by exocytosis
–  Releases membranes in milk, during virus budding, etc.
•  Mediates contacts with other cells, and external structures
–  Forms a variety of junctions
•  Provides the starting point for signal transduction pathways
•  Plays a central role during cell division, cell fusion, fertilization
The four layers of the PM from outside to
the inside
•
Extra cellular matrix
–  Assembled from proteins and
polysaccharides secreted by cells into
the intercellular space including
collagens, glycosaminoglycans
(GAG),
•
Glycochalyx
–  Glycoproteins, proteoglycans,
glycolipids (i.e. ‘glycoconjugates’)
attached to outside surface of the PM
•
Membrane bilayer
–  Lipids (cholesterol, phospholipids,
glycolipids), membrane proteins
(receptor proteins, channels, carriers,
pumps, enzymes, integrins, junctional
proteins, etc.)
•
Cell Cortex
–  Proteins associated with cytoplasmic
surface of PM; actin, kinases, adaptor
proteins, small GTPases, coat
proteins, lipid binding proteins, etc.
Plasma membrane domains
•  In many cell types, the plasma membrane has different domains and
different structural and functional specializations
•  These domains differ in composition and function
•  Many are permanent, some are transient
•  Many of them mediate interactions with different environments outside
stabilized by extracellular contacts (extracellular matrix, neighbouring
cells, etc.)
•  Others are stabilized by structures internally (examples: microtubules
in sperm tail and cilia, actin filaments in filopodia and microvilli)
•  Some are large (examples: apical and basolateral membrane domains
of epithelial cells)
•  Others are small (Examples: clathrin coated pits and caveolae)
Endocytosis: definitions!
Endocytosis: The internalization of substances and particles from the
extracellular space by invagination of the plasma membrane.!
•  Phagocytosis (large particles, specialized cells)
•  Pinocytosis (fluid and soluble macromolecules, most cells)
–  Fluid uptake (pinocytosis and macropinocytosis)
–  Receptor-mediated endocytosis
-  Caveolae/Raft mediated endocytosis
-------------------------•  Transcytosis (fluid and macromolecules across a cell between
apical and basolateral surfaces, epithelial and endothelial cells)!
Different types of endocytosis
Lipid!ra(!dependent!
pathways!
Conner and Schmid (2003)
What is in an endocytic vesicle?
•
•
•
•
Bulk!fluid!and!solutes!!
Bulk!membrane!components!
Selected!membrane!components!!
Receptor!bound!proteins!and!ligands!
NOTE:
Endocytosis is not only used to internalize particles, fluid
and ligands from the outside but also to:
- down regulate membrane receptors
- change lipid compositions
- etc.
In other words endocytosis controls plasma membrane
composition and thus fine-tunes function by selective
internalization of specific components.
Phagocytosis
Macrophages and neutrophils are phagocytosis specialists!
macrophage ‘eating’ red blood cells
1011 cells/day
Figure 13-46 Molecular Biology of the Cell (© Garland Science 2008)
neutrophil ‘eating’ a bacterium
Properties of phagocytosis 1!
•
Is induced by particle contact with cell surface receptors ( Fc receptors,
complement receptors, lectins, etc.)
•
Usually particles must be ‘opsonized’ for example by binding IgG so that they
can bind to the Fc receptors
•
Phagocytosis is mainly seen with ‘professional phagocytes’ i.e. macrophages,
polymorphonuclear lymphocytes, amoebae, slime molds, etc…
•
Macrophages also internalize remnants of cells that have undergone apoptosis
(programmed cell death), which expose phosphatidylserine (PS) on their surface
•
They do not internalize live cells that do not expose ‘eat-me’ signals on their
surface
•
Phagocytic vesicles can be large
•
Ingested particles are degraded in phago-lysosomes (fusion of phagosome with
lysosome)
•
Some bacteria manipulate the system and establish intracellular replication.
Properties of phagocytosis 2!
•
Requires binding at multiple sites to multiple
receptors around the entire particle (the ‘zipper’
mechanism). Limited space for fluid.
•
Mediated by elaborate signaling processes and actin
filament rearrangements on the cytosolic side
(triggered process)
•
A dramatic, transient modification of a PM domain follows
generating the phagocytic ‘cup’, and internalization the
phagocytic body, the ‘phagosome’
•
The ordered formation and consumption of
phosphatidylinositides guides sequential steps in the
process
–  formation of PI(4,5)P2 is needed for the formation of actin
containing membrane extensions – the pseudopods
–  conversion of PI(4,5)P2 by PI(3)kinase to PI(3,4,5)P3 drives
closure of the vacuole, and its internalization
Pinocytosis
•  Continuous process in virtually all cells
•  Vesicles are small and uniform
•  Surprisingly large volumes and membrane areas
involved
–  In macrophages, 25% of cell volume and 200% of cell
surface area pinocytosed every hour
•  Most of the volume and membrane are recycled back
to the plasma membrane
•  The coupled endocytosis/exocytosis ensures strict
control of cell surface area and cell volume
Endocytosis using clathrin
•
•
•
clathrin-coated pits occupy about 2% of PM
Short life-time: endocytosis is rapid (1 min)
rapidly shed their coat inside the cell
Here, internalizing lipoproteins in the chicken oocyte to form the egg yolk!
Figure 13-48 Molecular Biology of the Cell (© Garland Science 2008)
Clathrin-coated vesicle formation – a reminder!
Endocytosis signal located in the cytosolic tail
interacts with adaptins
AP2: plasma membrane to endosomes
Signal: (F/Y)xx(Y/F)
Endocytosis signal located in the cytosolic tail
interacts with CLASPs
Adap=ns!belong!to!the!clathrin>associated!sor=ng!proteins!(CLASPs)!
Adaptins connect clathrin
with membrane!
Cargo endocytosed by RME!
•  Nutrients, vitamins, and their carriers.
•  Hormones and growth factors.
•  Antigens to be processed and presented to the immune
system
•  Extracellular matrix components
•  Miscellaneous serum proteins
•  Asialo-glycoproteins (old serum glycoproteins devoid of sialic
acid in their glycans)
•  Receptor-bound viruses and toxins
•  And many other ligand receptor complexes!!
Receptor-mediated endocytosis along the clathrincoated vesicle pathway
Example 1: LDL receptor
Cholesterol uptake
Low-density lipoprotein particle
Figure 13-50, 13-51
Cholesterol uptake by LDL receptor
Normal and mutant
LDL receptors
Figure 13-51
Cholesterol uptake by LDL receptor
Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008)
Cholesterol uptake by LDL receptor
Figure 13-51
Receptor-mediated endocytosis along the clathrincoated vesicle pathway
Example 2: transferrin receptor
The transferrin cycle
Transferrin is the carrier for
Fe3+ in the body. It binds
the transferrin receptor at
the plasma membrane.
Cells endocytose it, and in
the endosome strip it off
its iron cargo.
The receptor with iron-free
transferrin recycles back
to the plasma membrane
Endocytosis using caveolae
•
•
•
•
Flask-shaped structure
Form from PM microdomains (lipid rafts)
lipid rafts are rich in cholesterol, glycosphingolipids, and GPI-anchored
proteins
May collect cargo by virtue of lipid composition
•  diameter: 70-100 nm
Figure 13-49 Molecular Biology of the Cell (© Garland Science 2008)
Endocytosis using caveolae
•  major integral membrane proteins: caveolins
•  major peripheral proteins: cavins
•  involved in transcytosis of serum components in endothelial cells
•  involved in cholesterol regulation, signal transduction, etc..
Caveolin
Makes a hairpin-loop in
the membrane.
Binds cholesterol
Figure 13-49 Molecular Biology of the Cell (© Garland Science 2008)
Caveolae: invaginating rafts!
Clathrin coated pit
Caveolae
The early endosome – a molecular sorting
station
An!isolated!early!endosome!
Figure 13-52 Molecular Biology of the Cell (© Garland Science 2008)
Receptors and ligands follow several
different intracellular pathways
!
Immunoglobulin trancytosis in epithelial cell!
E.g.:
Antibody uptake in
the gut of newborns
form the milk of the
mother
Figure 13-60 Molecular Biology of the Cell (© Garland Science 2008)
The early endosome – a molecular sorting
station
An!isolated!early!endosome!
Receptors and ligands follow several
different intracellular pathways
Figure 13-52 Molecular Biology of the Cell (© Garland Science 2008)
Down the road: the endocytic pathway
From early to late endosomes, and
lysosomes
Early endosomes mature to form late endosomes.
Early and late endosomes differ in their protein
composition, appearance and localization.
Many molecules are recycled. They are removed
by concentration in the tubular regions of early
endosomes. Loss of these tubules to recycling
pathways means that late endosomes mostly lack
tubules.
Lumen of endosomes is acidic (≈ pH 6).
They become increasingly acidic during maturation
mainly through the activity of the V-ATPase.
Figure 13-56 Molecular Biology of the Cell (© Garland Science 2008)
The vacuolar ATPase: The proton pump
responsible for acidification of
endosomes and lysosomes.
A multi-subunit trans-membrane complex that resembles
the F1F0 ATPase in mitochondria (the latter uses proton
flux through the membrane to synthesize ATP)
Vacuolar!ATPase!!! !
!
!
!F1F0!ATPase!!
Down the road: the endocytic pathway
Maturation of early to late endosomes
occurs through the formation of MVBs
(multivesicular bodies)
- molecules are sorted into smaller vesicles that
bud into the endosome lumen, forming lumenal
vesicles; this leads to the multivesicular
appearance of late endosomes and so they are
also known as multivesicular bodies.
-  MVBs move along microtubules to the the cell
center and continually shed recycling transport
vesicles to the PM
-  MVBs gradually convert into late endosomes
-  late endosomes no longer send vesicles to the
PM
- loss of Rab5 and binding of Rab7 marks the
transition from early to late endosomes.
Multivesicular bodies: on the way to the
lysosome
Figure 13-55 Molecular Biology of the Cell (© Garland Science 2008)
Multivesicular bodies: on the way to the
lysosome
Vesicles bud into the endosome
generating a ‘multivesicular body’. This
leads to selective degradation of
receptors and ligands in lysosomes.
The signal for uptake into intralumenal
vesicles are monoubiquitin tags added to
the cytosolic tail of receptors to be
degraded degraded in the lysosome.
!
Figure 13-57 Molecular Biology of the Cell (© Garland Science 2008)
Sorting into MVBs: the role of ubiquitin
and ESCRT complexes
Cargo!for!inclusion!is!recognized!and!sorted!by!the!
ESCRT!complexes!that!induce!the!forma=on!of!the!ILVs!
ubiquitin
Figure 13-58 Molecular Biology of the Cell (© Garland Science 2008)
Summary: Early to late
endosome maturation
1) pH drops from 6 or higher to less than 5.5
2) Intralumenal vesicles increase in number: ESCRT
complexes bind and PI(3)P is converted to PI
(3,5)P2
3) Movement to the perinuclear space via
microtubules
4) Most of the tubular extensions are lost
5) Rab5 GTPase is exchanged for Rab7
6) Capacity to fuse with early endosomes is lost and
with late endosome and lysosomes is gained
!
Summary (1)
•
Cells ingest fluid, molecules, and particles by endocytosis, in which
localized regions of the plasma membrane invaginate and pinch off to form
endocytic vesicles.
•
Many of the endocytosed molecules and particles eventually end up in
lysosomes, where they are degraded. Endocytosis occurs both
constitutively and as a triggered response to extracellular signals.
•
Many cell-surface receptors that bind specific extracellular macromolecules
become tagged with ubiquitin, which guides them into clathrin-coated pits.
As a result, these receptors and their ligands are efficiently internalized in
clathrin-coated vesicles, a process called receptor-mediated endocytosis.
The coated vesicles rapidly shed their clathrin coats and fuse with early
endosomes.
Summary (2)
•
Most of the ligands dissociate from their receptors in the acidic environment
of the endosome and eventually end up in lysosomes, while most of the
receptors are recycled via transport vesicles back to the cell surface for reuse.
•
But receptor-ligand complexes can follow other pathways from the
endosomal compartment.
–  In some cases, both the receptor and the ligand end up being degraded in lysosomes,
resulting in receptor down-regulation; in these cases, the ubiquitin-tagged receptors recruit
various ESCRT complexes, which drive the invagination and pinching-off endosomal
membrane vesicles to form multivesicular bodies.
–  In other cases, both receptor and ligand are transferred to a different plasma membrane
domain, causing the ligand to be released at a surface of the cell that differs from the
membrane where it originated, a process called transcytosis. The transcytosis pathway
involves recycling endosomes, where endocytosed plasma membrane proteins can be
stored until they are needed.
Plasma membrane (cont)
•  Mediates cell motility and determines cell size and shape
•  Forms the basis for structural and functional polarity of cells
•  Forms specialized structures such as cilia, microvilli, synapses
•  Provides synthesis of cell wall in fungi and plants
•  Interacts with invading pathogens and viruses, and participates in the
defense against them
•  By forming a ‘skin-tight’, ‘shrink wrapped’ surface, it helps to
determine the mechanical properties of cells
Bulk uptake is linearly dependent on concentration and not saturable
Receptor-mediated uptake is concentration dependent and saturable
A!fluid!phase!marker!!
Figure Q13-4 Molecular Biology of the Cell (© Garland Science 2008)
A!receptor>bound!ligand!!
Figure 13-61 Molecular Biology of the Cell (© Garland Science 2008)
Interes=ng!anima=ons!of!CME!
•  hNp://www.idi.harvard.edu/
inves=gators_research/mul=media/
kirchhausen_lab/!