lecture 8

Question 1

What is the Golgi apparatus?

Answer 1

• The golgi is a major site of carbohydrate synthesis as well as a sorting and dispatching station for products of the endoplasmic reticulum.
• a series of flattened sacs often referred to as cisternae
• it has two faces: cis face is the one closest to the ER, trans face closest to the plasma membrane
• has three set stacks: cis cisterna, medial cisterna, trans cisterna, and then the diffuse cis Golgi network and trans golgi network

Question 2

How is the Golgi apparatus functionally compartmentalised?

Answer 2

• each of the different cisternae have different enzymes which give them different functions:
  1. cis golgi network: phosphorylation of oligosaccharides on lysosomal proteins
  2. cis: removal of Man
  3. medial: removal of Man, addition of GlcNAc
  4. trans: addition of Gal, addition of NANA
  5. trans golgi network: sulfation of tyrosines and carbohydrates

Question 3

What two types of exocytosis are there?

Answer 3

some secretory vesicles continually traffic out to the membrane, bring things like newly synthesized plasma membrane lipids (need to be continally removed)

there is also regulated exocytosis: some vesicles are not continuously transported to the plasma membrane, will send it after receiving a certain signal e.g. insulin: transported from ER to golgi and packaged in a secretory vesicle. This vesicle doesn’t release contents until stimulated by hormone/neurotransmitter

Question 4

What is the pathway of endocytosis?

Answer 4

• starts out at plasma membrane and buds into the cytosol (clathrin coated pit)
• transported first to the early endosome: sorts the inward bound vesicles as opposed to outward which are sorted by TGN
• one of two options: either shunts vesicles onward to late endosome (microtubule-mediated transport) - multivesicular body transported along microtubule
• late endosome becomes endolysosome and eventually fuse with lysosome to form lysosome where products will be degraded.
• Or it can initiate a retrieval process. Some things such as receptors - e.g. for some signalling processes the receptor actually needs to be endocytosed, that receptor after the signalling event will actually be shunted back out to the plasma membrane by the early endosome

Question 5

Describe receptor-mediated endocytosis of LDL

Answer 5

A phospholipid monolayer filled with cholesteryl ester molecule. Cholesterol molecule in monolayer.
Low-density lipoprotein is bound by LDL receptor proteins –> forms clathrin coated pit and is endocytosed
naked vesicle fuses with early endosome but adaptor protein still part of membrane
protein buds off early endosome in transport vesicles and returns LDL receptors to plasma membrane while the LDL particle ends up in the lysosome to release free cholesterol

approx 1 in 500 individuals inherits one defective LDL receptor gene, and as a result, is likely to die prematurely from a heart attack caused by atherosclerosis - LDL receptor protein still concentrates cholesterol at the plasma membrane but it is not endocytosed. You cannot have mutations in both of these genes.

Question 6

How do exocytosis and endocytosis facilitate signalling across the neuronal synapse?

Answer 6

1. delivery of synaptic vesicle components to plasma membrane
2. endocytosis of synaptic vesicle components to form new synaptic vesicles directly
3. endocytosis of synaptic vesicle components and delivery to endosome
4. budding of synaptic vesicle from endosome
5. loading of neurotransmitter into synaptic vesicle
6. secretion of neurotransmitter by exocytosis in response to an action potential

Question 7

What must cells of metazoans do?

Answer 7

Associate to form organs

Question 8

What do the cytoskeletons of cells in epithelial tissue do?

Answer 8

they are linked - mechanical stresses are transmitted from cell to cell by cytoskeletal filaments anchored to cell-matrix and cell-cell adhesion sites

Question 9

What is the main stress-bearing component of connective tissue?

Answer 9

The extracellular matrix - directly bears mechanical stresses of tension and compression

Question 10

What are the four functional classes of cell junctions found in animal tissues?

Answer 10

• anchoring junctions
• -actin filament attachment sites: 1. cell-cell junctions (adherens junctions) 2. cell-matrix junctions (actin-linked cell-matrix adhesions)
• occluding junctions: 1. tight junctions (in vertebrates), 2. septate junctions (in invertebrates)
• channel-forming junctions: 1. gap junctions (animals), 2. plasmodesmata (in plants)
• signal-relaying junctions: 1. chemical synapses (in the nervous system), 2. immunological synapses (in the immune system), 3. transmembrane ligand-receptor cell-cell signaling contacts (Delta-Notch, ephrin-Eph, etc.). Anchoring, occluding, and channel-forming junctions can all have signaling functions in addition to their structural roles.

Question 11

What are tight junctions?

Answer 11

• specialised junctions of epithelial/endothelial cells
• also known as occluding junctions
• they facilitate transcellular transport
• they prevent molecules from diffusing between cells (i.e. the gap between two cells from apical to basal surface) so if a molecule wants to get from one side of the cell to the other it has to go through the cell and be recognised by specific receptors/channels etc
• tight junctions forms barriers to diffusion of: solutes - we can see this using dye solutions, and membrane proteins - this tells us that newly synthesised proteins are directed to specific regions of the cell membrane
• most apical of the junctions

Question 12

How are tight junctions formed?

Answer 12

• by a meshwork of sealing strands of transmembrane proteins
• points of tight contact are called focal connections - tight junctions consist of a series of focal connections
• the proteins that produce these are claudin and occludin - hydrophilic adhesion molecules
• one claudin binds one claudin in the other cell , one occludin one occludin
• they are associated with other cytoplasmic proteins such as JAM ( adhesion molecule)

Question 13

What are anchoring junctions?

Answer 13

• allow the cytoskeleton to adhere to ECM or other cells
• include: adherens junctions (connect to actin filaments), desmosomes, hemidesmosomes (connect to intermediate filaments)
• by anchoring adhesion molecules to the cytoskeleton it allows the cell to maintain robust cell-cell or cell-matrix adhesion: the cell changes behaviour under stress as opposed to simply ‘losing’ the adhesion molecules

Question 14

Of what are anchoring junctions composed?

Answer 14

consist of:

1. an intracellular plaque that attaches to the cytoskeleton
2. transmembrane proteins that bind to adjacent proteins on other cells/ECM

Question 15

What are adherens junctions?

Answer 15

• form a continuous belt below the tight junctions, the zonula adherens, in epithelial cells that bring actin filaments into alignment
• cadherins form the transmembrane linkages

Question 16

How can this belt of actin filaments be utilised?

Answer 16

• myosin motors can cause contraction of bundles of actin filaments in adhesion belts
• this results in cells to narrow at the apex
  1. sheet of epithelial cells
  2. invagination of epithelial sheet caused by an organised tightening of adhesion belts in selected regions of cell sheet
  3. epithelial tube pinches off from overlying sheet of of cells
  4. epithelial tube

this is how something like the neural tube forms in development

Question 17

How do cadherins (in adherens junctions) bind?

Answer 17

• in a homophilic manner
• monomeric adhesion
• binding is dependent upon calcium concentration: flexible hinge regions bind calcium molecules, in high calcium these flexible hinge regions become quite rigid, while without calcium molecules can flex and we don’t get good adhesion

Question 18

Where is another place where adherens junctions play an important developmental role?

Answer 18

Cells of the early mouse embryo stick together weakly
At the 8-cell stage they begin to express E-cadherin and as a result strongly adhere to one another
This is called compaction
One of the first important morphogenic events in embryogenesis

Question 19

How is adhesion important in tissue formation?

Answer 19

• homophilic adhesion and differential expression of classical cadherins can cause cell sorting
• e.g. E-cadherin vs N cadherin: causes cells of different organs to tightly stick together (in formation of neural tube e.g.) cells expressing different cadherins will spontaneously sort out in a dish
• even if you have cells of the same cadherin but expressing it at different levels - cells with high levels will adhere more strongly to one another and sort from those expressing low levels - cells of high level will be in the centre, low level on the outside - this is how you see organs with different tissue layers starting to form

Question 20

What links the classic cadherins to the cytoskeleton?

Answer 20

• catenins: p120- catenin, beta-catenin, alpha-catenin, plakoglobin (gamma catenin), vinculin, alpha-actinin

Question 21

What are the different classical cadherin molecules are where are they expressed?

Answer 21

• E-cadherin: many epithelia
• N-cadherin: neurons, heart, skeletal muscle, lens and fibroblasts
• P-cadherin: placenta, epidermis, breast epithelium
• VE-cadherin: endothelial cells

Question 22

What are the different non-classical cadherins and where is their main location?

Answer 22

• desmocollin: skin
• desmoglein: skin
• T-cadherin: neurons, muscle, heart
• Cadherin 23: inner ear, other epithelia
• Fat (in drosophila): epithelia and central nervous system
• Fat1 (in mammals): various epithelia and CNS
• alpha, beta and gamme-protocadherins: neurons
• Flamingo: sensory and some other epithelia

Question 23

What are desmosomes?

Answer 23

• “spot-weld” cells together to distribute tensile forces
• through desmosomes the intermediate filaments of adjacent cells are connected to form a continuous network of great strength
• the sides of cytokeratin filaments (intermediate filaments) interact with the cytoplasmic plaque that is attached to an adjacent cell via cadherin interactions
• the plaque is made from plakoglobin, plakophilin and desmoplakin

Question 24

How do hemidesmosomes compare to desmosomes?

Answer 24

• in contrast, hemidesmosomes attach to the ECM (via integrins) and to the ENDS of cytokeratin filaments
• they are chemically and functionally distinct from desmosomes
• hemidesmosomes utilise a specialised integrin (alpha 6 beta 4) to link cytokeratin filaments via plectin and dystonin anchor proteins to extracellular laminin
• integrins together with collagen XVII link through to laminin in the ECM

Question 25

What is epidermolysis bullosa?

Answer 25

- a genetic disease in which mutant keratin proteins do not form a stable cytokeratin network and skin cells blister - similar disease phenotypes are observed with mutations in components of hemidesmosomes

Question 26

What are Gap junctions?

Answer 26

- allow communication between cells, rather than having a structural role - they couple cells metabolically and electrically - formed of channels called connexons - interacting plasma membranes - gap between cells of 2-4nm - connexon composed of six subunits - channel 1.5nm in diameter - two connexons in register forming open channel between adjacent cells - each transmembrane subunit is called a connexin - can be heteromeric or homomeric, homotypic or heterotypic: will allow passage of different sorts of molecules based on structure - the channel in connexons allows passage of molecules up to 1000 daltons e.g. inorganic ions, sugars, amino acids, nucleotides, vitamins - each gap junction contains several hundred connexons - gap junctions are gated: different stimuli cause them to be open or closed e.g. low Ca2+ or high pH associated with opening the junction - pretreating a neuron with dopamine shuts off gap junctions with surrounding neurons: can be visualised with lucifer yellow

Question 27

What is the commonest cause of congenital deafness?

Answer 27

Cx26 mutations (a connexin protein)

Question 28

How do junctions contribute to cell polarity?

Answer 28

- one part of the multiple ways that cells can be polarised - polarity simply means that one part of the cell behaves differently to another - this is important in metazoan epithelial cells - but also in unicellular yeast cells - the cells on the right panel have been treated with mating factor from cells of the opposite mating type – they become polarised in order to direct the cell towards a potential fusion partner - polarity involves signals (extracellular or internal) and interactions with the cytoskeleton - and in some cases with cell junctions