Cells alive4: Vesicular transport: exocytosis/endocytosis

Overview the eukaryotic internal membrane systems and the transport that occurs between them • Discuss the principles of vesicular transport • Describe the ER and its role in exocytosis • Describe the Golgi apparatus and its role in exocytosis • Summarise constitutive and regulated exocytosis, giving examples • Explain how some neurotransmitters are released by exocytosis and the mechanism by which named bacterial toxins disrupt this process • Outline how cells take in large molecules and microorganisms by endocytosis, describe what fates await them, discuss how microorganisms are able to subvert this entry route to their own purposes

intro to organelles involved in vesicular transport

what is the endoplasmic reticulum (ER)?

Functions:

• Protein biosynthesis

• Lipid biosynthesis

• Intracellular Ca2+ store

Structure:

• The ER is made of tubules and sacs surrounded by membranes

• Tubules and sacs are

• developed from the nuclear outer membrane

• connected between each other

• Protrude into the cytoplasm

• The space encased enclosed by the membranes is called lumen or ER cysternal space

break down the functions of ER further

Protein synthesis

• ER and Golgi residents

• Secretory

• Transmembrane

• Lysosomal

Synthesis of lipids

• Cholesterol

• Sphingolipids

Can have cell specific functions:

• Synthesis of steroid hormones (eg male and

female gonads and adrenal glands

Enzymes; Detoxification from drugs (liver)

• Sarcoplasmic reticulum

Transitional ER: areas where vesicles bud from ER to go to the Golgi

ER post-translational modifications (PTM)

they can be modified by

disulphide bonds-stabilises the protein##

N-glycosylation-Attachment of multiple branched sugars to the amide nitrogen of an Asn

• Stabilise

• Protect from degradation

• Hold in the ER

• Serve as a signal for interaction with other

proteins

protein modifications of the endoplasmic reticulum

ER Post-translational modifications:

1.Formation of disulphide bonds

2.Proper folding

3.Addition and processing of carbohydrates (N-linked glycosylation-Asn)

4.Specific proteolytic cleavages

5.Assembly into multimeric proteins

the completed protein is then sent to the Golgi

what is the structure of the golgi apparatus?

located near the nucleus/close to the centrosome

divided into cisternae - they constantly communicate with each other through vesicles

what are the functions of the golgi?

• Carbohydrate synthesis

• Post-translational modification of proteins and lipids

• Glycosylation (O-linked)

• Sugar added to an oxygen atom of Ser or Thr

• Phosphorylation

• Sulphation

• Sorting and dispatching station for products of the ER

The Golgi is the sorting office of the cell in the secretory pathway

(exocytosis) and therefore, tends to be prominent in secretory cells like this intestinal goblet cell

what are endosomes?

the other ‘sorting office’

EARLY ENDOSOME

• They reside under the plasma membrane

• It “matures” into the late endosome

• By fusing with each other

• By fusion with a late endosome

LATE ENDOSOME

• Located near the nucleus

• Sorting compartment in the endocytic pathway (endocytosis)

• Receptors recycled to the membrane

• Receptors/ligand addressed to the lysosome

• Receptors/ligand addressed to another domain of the plasma membrane

• Acidic environment (ATP-driven H+ pump)

what are lysosomes?

Recycling function

• Protective

- membrane keeps enzymes out of the cytosol

- acid hydrolases don’t work at cellular pH (7.2)

• ATP-driven pump that pumps H+ into the lysosome keeping the pH very low

• Digestion products diffuse or are pumped out of the lysosome

summarise the functions of each organelle

give me an overview of vesicular transport

• Association of “cargo” with the area of the donor membrane which will give rise to the vesicle

• Membrane distorted to form a bud

• Detachment of bud to form a vesicle

• Movement of vesicle containing cargo across cell to vicinity of recipient membrane

• Recognition of & binding to recipient membrane

• Fusion of vesicle with recipient membrane & release of cargo

how are vesicles formed?

1 - Cargo molecules bind to transmembrane receptors

2 - Curved coat proteins (COP I, COP II or clathrin) recruited & distort membrane

3 - Vesicle released by dynamin

4 - Vesicle rapidly uncoated

what is vesicle targetting?

vesicles are fussy with what they fuse with

• Vesicles have surface markers: identify origin & cargo

• Wrap around one another forming trans-SNARE complex: locks membranes together (docking) & mediates membrane fusion (see later)

• Rab proteins direct the vesicles to their specific ‘spots’ on the membrane

• >20 SNARE proteins work in pairs: each vesicular v-SNARE has a complimentary target

membrane t-SNARE

• Complementary receptors displayed on target membranes

how does transport between the ER and golgi work?

1. cargo is recruited to exit sites in the ER- via receptor if soluble cargo

2. COPII recruited to cause budding

3. vesicles rapidly shed their coat (COPII released from surface)

4. vesicles undergo homotypic (like-to-like) fusion mediated by SNAREs

5. newly formed vesicular tubular clusters (VTC) moved along microtubules by dyneins (motor proteins)

6. …to the Golgi where they fuse and deliver their contents

what is the golgi-ER retrieval mechanism?

cargo release is mediated by a decrease in pH

various proteins need to be retrieved: escaping ER proteins, receptor proteins

have signal sequences (KDEL sequence)

vesicles coated in COPI bud from the VTC and Golgi, are uncoated and transported back to the ER

retrieval pathway can be hijacked by bacterial toxins to gain entry to cytoplasm

how do cells communicate with the extracellular environment?

Endocytosis (import of material from outside)

• Pinocytosis

• Phagocytosis

Exocytosis (export of material from inside)

what are the 2 pathways for exocytosis?

Constitutive secretory pathway

TGN → plasma membrane

Protein do not require a signal to be secreted through this pathway

Regulated secretory pathway

Specialised secretory cells

• hormones

• neurotransmitters

• digestive enzymes

Prior to fusion, vesicle contents may be concentrated or processed

What are bacterial toxins and exocytosis

some non-protein molecules are released by exocytosis e.g. neurotransmitters like acetyl choline and GABA

• Docking

• Priming

• Ca2+ intake-action potential

• Firing

what is synaptic signalling susceptible to?

interference from toxins

Tetanus and Botulinum toxin are proteases which cleave trans-SNARE complexes formed

when vesicles dock at the synaptic membrane

• Therefore the tSNAREs needed for the next battery of vesicles to dock are destroyed

• The result is that synaptic transmission is blocked

• These toxins are highly specific, entering only certain neurons

• The outcome can be fata

what is botox?

it is actually a poison that is now a drug - a popular non-surgical cosmetic treatment

it is a neurotoxin derived from Clostridium botulinum (an organism found in the natural environment, where it is largely inactive)

used to reduce fine lines and wrinkles by paralysing the underlying muscles

also used to treat excessive sweating, migraines, muscular disorders, etc.

botulism (botulinum toxin-illness) can cause respiratory failure

• 1g of botulinum toxin can kill > 1m people

• 2kg is enough for the entire population

• infant botulism and honey

endocytosis

Intake of molecules from the extracellular space and from the membrane

• Nutrients

• Recycling

Balance between endo- and exocytosis

what is pinocytosis?

a type of endocytosis (one of the 2 ways)

recycling of membrane and non-specific uptake

clathrin-coated pits

receptor mediated endocytosis - e.g. LDL-cholesterol

what is phagocytosis?

the other form of endocytosis

P H A G O C Y T O S I S Specialised white blood cells

1. Interaction receptor-phagocytosis trigger (eg Ab- Epitope)

2. Rearrangement of the cytoskeleton: pseudopods formation

3. Formation of the phagosomes

4. Fusion of the phagosome with the lysosome

It depends on a balance between positive and negative stimuli

what happens to endocytosed vesicles?

Fuse with early endosomes: a sorting site for endocytosed molecules

Several fates then await….

1. Recycling

Membrane & many receptors sent to “recycling endosome”

Vesicles return to plasma membrane

e.g. LDL receptor releases LDL in acidic early endosome

LDL receptor returns to PM for more cargo

2. Transcytosis

Vesicles return to different part of PM; transports material across cell e.g. maternal antibodies carried across gut epithelium by transcytosis

3. Degradation

Cargoes (& some receptors) sent to late endosomes (mechanism not precisely known) which mature into lysosomes

Macromolecules degraded & their components used to make new molecules

e.g. LDL sent to late endosomes and cholesterol released by degradation of the rest of the particle in the lysosom

summarise vesicular transport

Intracellular (organelle to organelle) communication is mediated by VESICLES

• The process is mediated by several molecules

• Clathrin/COPI/COP2 mediate recruitment of the molecules to transport

• The fusion of vesicles is mediated by SNARE complexes

Extracellular communication (endocytosis and exocytosis) is mediated by VESICLES

• The content uptaken from the ECE through vesicles can either be

• Directed to a specific location in the cell

• Recycled

• Fuse to another location of the plasma membrane

• Degradation