Exocytosis & Endocytosis

Question 1

What is endocytosis

Answer 1

Transport of macromolecules made in the cell from the trans golgi network to the plasma membrane/extracellular space via vesicle and membrane fusion
used to release material, increase plasma membrane, insertion

Question 2

2 types of Exocytosis

Answer 2

1. constitutive exocytosis (occurs naturally and constantly)

2. regulated exocytosis (secretory vesicles wait near plasma membrane until signal)

Question 3

Dense core secretory vesicles

Answer 3

• This term is often used to refer to secretory vesicles
• It makes sense if you think about how a secretory vesicle would appear under an electron microscope
• “electron dense” areas of a sample (usually made dense by staining techniques) will be observed as dark areas in the image
• Secretory vesicles will have dense cores due to the cargo they carry

Question 4

Maturation of secretory vesicles

Answer 4

• Immature secretory vesicles: bud off from the trans golgi network and still resemble the structure of trans golgi cisternae
• During maturation: several immature secretory vesicles fuse together, cargo becomes concentrated increased concentration of V-type ATPases in the vesicular membrane results in protons being pumped in (acidification), proteolysis processing
• Final mature secretory vesicles: very densely packed allowing exocytosis of large amounts at once

Question 5

Proteolysis and secretory vesicles

Answer 5

• Proteolysis is the breakdown (hydrolysis) of proteins into smaller peptides
• This process allows precursor proteins to be converted into active, mature proteins
• Precursor proteins used in order to protect the cell from damage
• short proteins may not have enough room to include the necessary packaging signals
• Same precursor can synthesize different proteins depending on the modifications done after synthesis

Question 6

‘Priming’ of secretory vesicles

Answer 6

SNARE hypothesis: the idea that one membrane contains a v-SNARE and the other membrane a t-SNARE

• An interaction between these proteins generates a 4 alpha helical bundle complex – this exerts a force to overcome the energy barrier needed for fusion
• During the ‘priming’ step, the SNAREs are already partially paired but the helices are not completely wound yet

Question 7

Cell polarization

Answer 7

• Polarization requires the membrane to contain two or more distinct domains, in terms of molecular composition and/or function
• Molecules can be directed to a specific part of the membrane using sorting signals
• Or, in rarer cases, random delivery followed by selective stabilization or elimination (after arrival at plasma membrane), ie. components delivered indiscriminately but are selectively eliminated

Question 8

Cytotoxic Granule Exocytosis

Answer 8

• Human cytotoxic (killer) T cells form immunological synapses with cancer cells
• Here, the killer T cells exocytose granules containing toxic molecules such as perforin and cytolysin

Question 9

Example of vesicle priming - Neurotransmitters

Answer 9

• Nerve cells contain both dense core vesicles and synaptic vesicles containing neurotransmitters
• the speed of neurotransmitter release indicates that after vesicles dock at the presynaptic membrane, they are primed to prepare for rapid fusion
• complexin proteins freeze the SNARE complexes into a partly wound state
• signal releases this protein to allow fusion and component dispersal

Question 10

Endocytosis

Answer 10

process by which material enters a cell

can be quick

Question 11

3 types of endocytosis

Answer 11

1. Pinocytosis = fluid phase
2. Phagocytosis = large material
3. Receptor mediated endocytosis

Question 12

Pinocytosis

Answer 12

Small pinocytotic vesicles
Usually continually made - can remove damaged membrane sections
Clathrin coated vs caveolae vesicles
- clathrin coated pits in membranes invaginating in and pinching off before fusing with early endosome
- caveolae can also form vesicles via lipid rafts
Dynamin encourages vesicle pinching off
Caveolae can fuse with endosomes or transcytose
Caveolins don’t need to be released unlike clathrin coats

Question 13

Phagocytosis

Answer 13

‘Professional phagocytes’ = macrophages and neutrophils
Is a triggered process - cell surface receptors but no clathrin coated pits
- EG. apoptosis triggered by loss of membrane asymmetry

Question 14

Receptor Mediated Endocytosis

Answer 14

Specific macromolecules triggers vesicle formation
Can concentrate ligand thousand fold without taking up lots of extracellular fluid
Uses clathrin coated pits and vesicles
Increases efficiency of uptake

Question 15

3 fates of receptor mediated endocytosed proteins

Answer 15

1. recycling
2. transcytosis
3. degradation

Question 16

Cholesterol Uptake

Answer 16

Most cholesterol moved in blood in LDL
Protein and phospholipid later surrounds cholesterol
Adaptin protein on cell membrane recognises protein and binds to LDL and recruits clathrin
Clathrin coat causes invagination of vesicle
Fusion with endosome (low pH) and cargo released
vesicles are recycled and LDL is digested by hydrolytic enzymes
- Clinical relevance: some cells can’t take up cholesterol
Result - too high blood cholesterol = coronary artery disease
- Uptake is mediated by negative feedback  too much free cholesterol in the cell results in downregulation of cholesterol synthesis and LDL receptor expression

Question 17

Iron Uptake

Answer 17

Transferrin + iron binds to cell receptor is endocytosed
in endosomes, the iron is released
receptor and transferrin is recycled
transferrin is released in neutral pH of extracellular fluid - can pick up more iron
- repeating cycle

Question 18

Macropinocytosis

Answer 18

Triggered by growth factors (specific cargos), cell ruffles, macropinosomes
Ligands activate a pathway forming cell-surface protrusions
Ruffle extends from the membrane, folds over, and encloses cargo
This process can be exploited by pathogens to enter a cell
Clathrin independent endocytotic pathway

Question 19

Endosomes

Answer 19

• Endosome maturation happens throughout the endocytic pathway
• Early endosome  late endosome (or recycling endosome)  endolysosome
• Remember, it is not a simple linear pathway. Endosomes are dynamic organelles with vesicles travelling bidirectionally between early/late endosomes and the golgi network
  Early endosomes have tubules (membrane surface) and vacuolar domains (cell volume)
  During maturation, the tubular portions shrink and the vacuolar portions are retained/transformed
  Maturing vesicles (multivesicular bodies) migrate inwards (fuse into lysosomes)
• v-type ATPase pumps H ions in and other proteins change the cytosolic face of the membrane