Membrane Trafficking

7/28/2019 Membrane Trafficking

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Intracellular

Compartmentalization


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The major intracellular compartments

of an animal cell


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Compartmentalization of Cells

Membranes

Partition cell Important cellular functions

Impermeable to most hydrophobic molecules

contain transport proteins to import and export specific molecules

Mechanism for importing and incorporating organelle specific proteins thatdefine major organelles


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Major Organelles

Nucleus

Cytosol

ER

Golgi Apparatus

Mitochondria and Chloroplast

Lysosomes

Endosomes

Peroxisomes


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Relative Volumes Occupied by the Major

Intracellular Compartments

INTRACELLULAR COMPARTMENT PERCENTAGE OF TOTAL

CELL VOLUME

Cytosol 54

Mitochondria 22

Rough ER cisternae 9

Smooth ER cisternae plus Golgi

cisternae

6

Nucleus 6

Peroxisomes 1

Lysosomes 1

Endosomes 1


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Four distinct families

1) the nucleus and the cytosol, which communicate through

nuclear pore complexes and are thus topologically

continuous (although functionally distinct);

2) all organelles that function in the secretory and endocyticpathways, including the ER, Golgi apparatus, endosomes,

lysosomes, the numerous classes of transport

intermediates;

3) the mitochondria;

4) the plastids (in plants only).


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Topology governed by evolutionary

origins

Organelles arising from pinching off of PM have interior equivalent to

exterior of cell


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Development of Nucleus and ER

Topology governed by evolutionary origins

Invagination of pm creates organelles such as nucleus that are

topologically equivalent to cytosol and communicate via pores


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Development of Mitochondria

Endosymbiosis of mitochondria and plastids creates double

membrane organelle which have their own genome.


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Development of plastids


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3 Types of Transport Mechanisms

1. Gated Transport:

gated channels

topologically equivalent spaces

2. Transmembrane Transport:

protein translocators

topologically distinct space

3. Vesicular transport:

membrane enclosed intermediates

topologically equivalent spaces


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Membrane Trafficking


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Endo-membrane system

General concepts of vesicle-mediated traffic

Sequence of events beginning with ER and ending

at PM Details of Golgi function

Golgi to PM pathway

The Endocytotic Pathway

The Exocytotic Pathway

Membrane Trafficking


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Endomembrane Network The endomembrane system is a network of organelles

in eukaryotic cells Exocytosis begins in the endoplasmic reticulum

The Golgi apparatus modifies and sorts proteins in theexocytic pathway

Exocytosis ends at the plasma membrane

Endocytosis begins at the plasma membrane Endocytosis ends at the lysosome


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The endomembrane system is a network of

organelles in eukaryotic cells

The endomembrane system is a set of

interconnected organelles that readily exchange

materials. The primary functions of the endomembrane

system are to control the export (exocytosis) and

import (endocytosis) of materials to/from the

extracellular space.


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The endomembrane system is a network of

organelles in eukaryotic cells

Membrane-bound compartments called vesicles

shuttle between organelles in the endo-membrane

system and are responsible for carrying material

from one organelle to another.

The creation, transport, targeting, and fusion of

vesicles occurs in nine steps.


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The endomembrane system controls

molecular transport in/out of cell

Endocytic and

exocytic pathways

Endomembrane

system

The endocytic and exocytic pathways.


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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 ofmembrane-limited vesicles

Endocytosis

O2

O2

cytoplasm

Ca2

Y

Y

Y

extracellular

Ca2


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Membrane Trafficking: Vesicular Transport

Transport large particles or fluid droplets through membrane in vesicles

uses ATP

Large packets of substances and engulfed cells move across the plasma membraneby processes of endocytosis and exocytosis

Exocytosistransport out of cell

Endocytosis

transport into cell phagocytosisengulfing large particles

pinocytosistaking in fluid droplets

receptor mediated endocytosistaking in specific molecules bound to

receptors

Membrane lipids and proteins move to and from the plasma membrane duringthese processes


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Vesicular

Transport

RER to cis Golgi

Modified in Golgi (glycosylation,

phosphorylation)

Sorted at trans Golgi network into

Lysosomal (endocytosis)

Regulated (exocytosis)

constitutive (exocytosis)


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Vesicles shuttle material between organelles in the endomembranesystem

Donor/acceptor

compartments

Vesicle-mediated

transport

Exocytosis,endocytosis, and

fusion of vesicles

In vesicle-mediated transport, a

membrane-bound vesicle buds from

one compartment and fuses with

another.


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The Golgi apparatus plays a central role in vesicular traffic

within cells.


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The Golgi apparatus modifies and sorts

proteins in the exocytic pathway

The Golgi apparatus is organized into discretecompartments called cisternae. The cisternae arestacked on top of one another, and are classifiedas cis, medial, or trans according to their relativelocation within the overall Golgi structure.

Golgi-resident proteins are primarily responsiblefor modifying proteins undergoing exocytosis.They are retained in the Golgi apparatus bytransmembrane Golgi retention sequences.


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The Golgi apparatus modifies and sorts

proteins in the exocytic pathway

The extreme ends of the Golgi apparatus are

elaborated into long, tubular structures called the

cis Golgi network and trans Golgi network.

Both Golgi networks sort proteins into vesiclestargeted to different locations. The trans Golgi

network is especially effective at sorting a large

number of proteins into many distinct vesicle

types.


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Proteins exiting the ER join the Golgi apparatus at the cis Golgi network. The Golgi

apparatus consists of a collection of stacked compartments.


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Pathway Through Golgi Apparatus

1. Molecules come in vesicles

2. Vesicles fuse with Golgi membrane

3. Molecules may be modified by Golgi

4. Molecules pinched-off in separate vesicle

5. Vesicle leaves Golgi apparatus

6. Vesicles may combine with plasma membrane to secretecontents


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The Golgi Apparatus

has two major

functions:

1. Modifies the N-linked

oligosaccharides and adds O-

linked oligosaccharides.

2. Sorts proteins so that whenthey exit the Trans Golgi

Network (TGN), they are

delivered to the correct

destination.


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Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various

Golgi compartments.

1. Sorting in TGN

2. Protection from protease digestion

3. Cell to cell adhesion via selectins


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Once proteins that dont normally reside in the ER are properly folded,

they are transported to the golgi apparatus.


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Three main types of coated vesicles

Each type of vesicle is named according to its primary coatproteins:

- Clathrin vesicles transport proteins from plasma

membrane or the trans-Golgi network to late

endosome.

- COPI vesicles transport proteins in between Golgicisternae and from the cis-Golgi back to the rough ER.

- COPII vesicles transport proteins from the rough ER to

the Golgi.


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The three main types of coats involved in vesicle-mediated transport :


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Model for the formation

of a clathrin-coated pitand the selective

incorporation of integral

membrane proteins into

clathrin-coated vesicles

COPII t d i l t t i th i l t b l l t ( t ) t th i G l i t k


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COPII coated vesicles transport via the vesicular tubular cluster (vtc) to the cis-Golgi network.

The protein coating is removed

and the vesicles fuse with each

other to form the vtc.

The vtc is motored by kinesin

(motor protein) along

microtubules that function like

engine on rail tracks.

The vtc fuses with the cis-Golgi

network.

COPI transports vesicles back toER

i i h i h h h l i


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It is uncertain how proteins move through the Golgi apparatus.

Stationary compartments

with vesicles transportingbetween compartments.

Large moving compartments

that mature into the TGN, and

return enzymes to trailing

compartments by retrieval

vesicles.


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The trans Golgi network (TGN) sorts

proteins exiting the Golgi apparatus

Dynamin uses GTP to regulate scission of a vesicle from a donor compartment.


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TGN sorting mechanisms are complex

Many different cargo selection mechanisms

PTM (post-translational modifications)

Protein aggregation

Signal receptor

Lipid raft


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Budding of vesicles at the TGN likely occurs

by several different mechanisms

3 different mechanisms have been proposed

Curvature-inducing proteins

Modification of membrane phospholipids

Phospholipid asymmetry

Vesicle mediated transport 9 steps


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Vesicle-mediated transport - 9 steps

1) Cargo selection

2) Budding

3) Scission

4) Uncoating

5) Transport

6) Tethering7) Docking

8) Fusion

9) Disassembly

Vesicle-mediated transport occurs in 9 steps.

E d t i d E t i


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Endocytosis and Exocytosis

Endocytosis: a process of uptake of extracellular material by

engulfing it within cell, including receptor-mediated endocytosis,

phagocytosisandpinocytosis.

Exocytosis: a process of release of intracellular molecules (such as

hormones, secretory proteins) contained within a membrane-

bounded vesicle by fusion of the vesicle with its plasma membrane.


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Endocytosis

Small region of the plasma membrane invaginates toform membrane-limited vesicles.

Internalized molecules retain topology (lumen =extracellular)

Cargo can be specifically selected (receptor)

Destination of cargo can be controlled;


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Some of the functions of endocytosis

Nutrient uptake

Plasma membrane protein regulation and/or

degradation

Synaptic vesicle recycling

Trans-cellular signaling

Exploitation: virus and toxin entry into cells


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Three Pathways of Endocytosis

Phagocytosis

Pseudopods engulf target particle and merge as avesicle, which fuses with a lysosome in the cell

Pinocytosis

Extracellular fluid is captured in a vesicle andbrought into the cell;

Receptor-mediated endocytosis

Specific molecules bind to surface receptors,which are then enclosed in an endocytic vesicle


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Phagocytosis: engulfment of particles

Ingestion of microbes or particles by a cell

Transports specific substance Relatively large in size (~.75micro m)


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Phagocytosis in Multicellularorganisms

occurs only in specialized cells likemacrophages, dendritic cells

andneutrophils.

capture and destroy pathogens and particulate antigens

essential component of the immune response

Fc- and complement-receptor mediated phagocytosis

named for binding specificity for antibody tail region called Fc

(Fragment, crystallizable)


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Phagocytosis in Macrophage: The cells receptors in the plasma membrane

enable them to recognize their targets. For example, macrophages have

receptor that recognizes phosphatidylserine which becomes exposed on the

surface of dead cells.

Pinocytosis: internalization of fluid


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Pinocytosis: internalization of fluid

Generated at sites of ruffling at the plasma membrane

Non-specific in substance it transports

Classified as macropinocytosis (vesicles > 1 mm in diameter) andmicropinocytosis (vesicles < 200 nm in diameter)


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Receptor - Mediated Endocytosis :

A selective process

Involves formation of vesicles at surface of membrane

Vesicles contain receptors on their membrane

Receptors bound to specific target molecule

Clathrin-coated vesicle in cytoplasm

uptake of LDL from bloodstream

If receptors are lacking, LDLs accumulate andhypercholesterolemia develops


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Receptor-Mediated Endocytosis

Nutrient Uptake

(LDL,transferrin, etc.)

Membrane Recycling

Membrane Protein

Recycling

Antigen Uptake

Synaptic Vesicle

Recycling

Signaling Receptor

Down-Regulation


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Receptor Mediated Endocytosis


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Receptor-Adaptin Association Nucleates Pit Formation

Receptors Bind Cargo.

Clathrin Adaptins [AP1(Golgi) or AP2(PM)] bind to Receptor Tail Sequences.

Coated Pits Form and Pinch Off into Coated Vesicles

GTP h d l i b d i i i d f


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GTP hydrolysis by dynamin is required forpinching off of clathrin-coated vesicles

Dynamin-GTP forms a collar

around the neck of a coated

Pit.

GTP hydrolysis by dynamin

is required for pinching off.


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Endocytosis begins at the plasma membrane

The onset of endocytosis is most often indicatedby the clustering of cargo receptors on the

plasma membrane, accompanied by the

assembly of a clathrin coat on the cytosolic face

of the cluster. In micrographs, this structure

resembles a pit in the membrane, so it is often

called a coated pit.

Coated pits complete the nine steps of vesicletransport and deliver the vesicle to an organelle

called the endosome.

Clathrin stabilizes the formation of vesicles


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Clathrin stabilizes the formation of vesicles

The role of clathrin in endocytosis.


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Caveolin mediated endocytosis

Caveolae are 50-100 nm invaginations on the cells surface

Caveolin, a membrane protein, is the coat protein of caveolae

Undergo endocytosis in response to a signal (ex. SV40binding) in a cholesterol- and dynamin-dependent fashion

Internalized caveolae recruit actin to form comet tails

Upon internalization caveolae are delivered to novelendosomal compartments known as caveosomes

Th d t t i i th d ti


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The endosome sorts proteins in the endocytic

pathway

The endosome is formed by the fusion of endocytic

vesicles with specific vesicles that bud from the TGN.

The endosome sorts materials arriving from the

plasma membrane; cargo receptors are recycled tothe plasma membrane, while cargo remains in the

endosome.

The lumen of the endosome is slightly acidic relative

to the extracellular space, and this acidity is key tothe sorting mechanism.

This sorting mechanism is very different from the

sorting mechanisms used in the Golgi apparatus.

Th d i bdi id d i t l d


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The endosome is subdivided into early and

late compartments

Specific interactingmolecules ensure correctvesicles fuse withvesicles from TGN or

early endosomes

Proton pump proteinsplay central role insorting/activation ofendosomal contents Maturation of endosomes to form lysosomes.

The endocytic pathway is divided into the early


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The endocytic pathway is divided into the early

endosomes and late endosomes pathway.

Materials in the early endosomes are sorted:

Integral membrane proteins are shipped back to the

membrane;

Other dissolved materials and bound ligands retained.

Dissociation of internalized ligand-receptor complexs in

the late endosomes: Molecules that reach the late

endosomes are moved to lysosomes.


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Endocytosis ends at the lysosome

Complete degradation of endocytosed materials

takes place in the lysosome.

The lysosome is likely generated from the endosome

in several ways, and requires fusing a vesicle fromthe TGN that contains essential proton pump proteins

and digestive enzymes with the endosome.

Once digested, the cargo building blocks (sugars,

nucleosides, amino acids, etc.) are transported intothe cytosol for reuse.

Lysosomes: membranous organelles filled with


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Lysosomes: membranous organelles filled withdigestive enzymes

Breakdown endocytosed

materials

Through phagocytosis or receptor

mediated endocytosis

Breakdown old organelles(residual body)

Autophagy by autophagosome

One ultimate destination of some proteins that arrive in the TGN is the


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lysosome. These proteins include acid hydrolases.

Lysosomes are like the stomach ofthe cell. They are organelles

surrounded by a single membrane

and filled with enzymes called acid

hydrolases that digest (degrade) a

variety of macromolecules.

A vacuolar H+ ATPase pumps

protons into the lysosome causing

the pH to be ~5.

Digestion of cargo molecules in the

lysosome.

The macromolecules that are degraded in the lysosome arrive by endocytosis -

phagocytosis or autophagy


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phagocytosis or autophagy.

Phagocytosis vs Autophagy


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Phagocytosis vs. Autophagy

Phagocytosis

Autophagylysosomes


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pH is used in 3 ways to control endocytosis

1) the acidic environment in endosomes helps sort

cargo from receptors

2) the relatively neutral pH of the ER and Golgi

apparatus keeps the hydrolytic enzymes fromdigesting these organelles

3) the enzymes requirement for a strong acid

environment protects the endomembrane system

from digesting itself

Vesicular Transport: Exocytosis


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Vesicular Transport: Exocytosis

Secreting material or replacement of plasmamembrane

Exocytosis


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y

Vesicle moves to cell surface

Membrane of vesicle fuses

Materials expelled orCell

discharges material

Reverse of endocytosis

Where do newly synthesized membrane andsecretory proteins need to go and how do they get

there? Secretion (constitutive and regulated) PM protein delivery (polarized and non-polarized cells)

Lysosomal targeting

Constitutive (un-regulated) and regulated secretion


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Constitutive (un regulated) and regulated secretion

Constitutive secretion/ exocytosis of


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Constitutive secretion/ exocytosis of

plasma membrane proteins

Delivered via membranevesicles directly from the TGNto the cell surface

Share same vesicles asconstitutively secreted proteins

Remarkably little is knownabout how plasma membraneproteins are sorted intosecretory vesicles

May be more than one class ofcarrier vesicles

A model protein for studying the

secretory pathway (shown here tagged

with GFP)

R l t d ti


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Regulated secretion

Occurs in endocrine,exocrine and neuronalcells

Insulin secretion in

pancreatic b-cells Trypsinogen secretion

in pancreatic acinarcells

Exocytosis occurs inresponse to a trigger(ex. Ca2+)

2 mechanisms for controlling the final steps


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2 mechanisms for controlling the final steps

of exocytosis

Constitutive secretion

constant

Regulated secretion

Controlled bysignaling proteins

Secretory vesicles

(zymogen granules) Condensing vacuole

Figure 09.11: Transmission electronmicrograph of clathrin-coated pits and

vesicles at the oocyte surface.


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Exocytosis ends at the plasma membrane

Cells regulate the last stage of exocytosis(fusion) for most exocytic vesicles, to control

when and how much material is released into the

extracellular space and to control the delivery of

membrane-associated proteins to the plasmamembrane.

Controlled secretion is also called regulated

secretion, and is under the control of signalingpathways.

Transcytosis : provides a way to deliver proteins across epithelium


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Transcytosis : provides a way to deliver proteins across epithelium.

Transport of antibodies in

milk across the gut

epithelium of baby rats.

Acidic pH of the gut favorassociation of antibody with

Fc receptor whereas the

neutral pH of the

extracellular fluid favors

dissociation.


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Transcytosis: a closer look


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Transcytosis: a closer look Transcytosis: transport of

macromolecular cargo from

one side of the cell to theother

Transcytosis is also utilized inthe biosynthetic trafficking ofsome PM proteins

pIgA-receptor is a model forstudying transcytosis Contains sorting information in

its cytoplasmic tail

pIgA is secreted into the the gutlumen, bile and milk as part of

the mucosal immune responsepIgA

pIgA-R

synthesizes IgA

lumen

Blood/interstitial

Membrane Trafficking

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