AH Lectures 1-5 Flashcards
What is exocytosis?
Transport of proteins and lipids that are made within the cell to the exterior of the cell –> Via vesicle membrane fusion with P.M.
Note –> When vesicles are released from elsewhere in the cell this is called vesicle budding rather than exocytosis
What two categories can exocytosis be divided into? Outline how each of the two types work.
- One type happens by default without signals –> constitutive exocytosis –> happen all the time without signal (almost by default) –> E.g. mucus in the lung lining.
- The other requires signals to occur –> non-constitutive exocytosis –> Pool of vesicles wait by the membrane and only undergo fusion in response to a signal –> i.e. synaptic vesicle fusion between two nerve cells –> this process is Ca2+ dependent –> It interacts with PM proteins to trigger snare entwining –> Reserve pool waiting behind those for the next impulse.
Regulated secretory vesicles are warped along microtubules using motor proteins –> Primed e.g. snares touching and just waiting for a calcium influx
Number two –> Especially relevant for specialised secretory cells e.g. hormones (insulin), digestive enzymes, neurotransmitters
What is the function of exocytosis?
Functions:
- Releasing proteins into the extracellular space
Whatever is in the lumen of the vesicle will be exposed to the exterior to the plasma membrane when it fuses.
- Insertion of membrane proteins into P.M –> proteins within the membrane of the vesicle will add to the P.M.
- Vesicles fusing with P.M is also important for cell growth –> when a cell grows the membrane has to get bigger so exocytosis can insert more lipids and proteins
Explain the process of vesicle formation.
The process is not well understood –> Domains in lipid P.M components are thought to be involved e.g. lipid rafts used to concentrate proteins.
However, there are a couple of key processes that tend to occur before exocytosis:
- Vesicle Maturation: removal of clathrin as it can stop fusion to PM, this is recycled then reused elsewhere.
- Vesicles are constantly taken back –> to replenish Golgi –> otherwise it will shrink.
- Cargo within vesicle tends to be highly concentrated –> maximise transport
- Active processing proteins –> Cleavage can activate the protein just before it is released –> E.g. cleavage of pro-peptides at the N terminal.
- Kiss+run –> when a little bit of fusion occurs and vesicle content is released –> the vesicle can then be used again –> Key in the immune system, especially mast cells.
Can exocytosis be localised? How is this achieved?
Exocytosis can be localised or all-around PM
Polarised cells have a direction –> Mainly controlled by cell adhesion blocking off parts of the cell (preventing exocytosis in particular regions) or cytoskeleton leading vesicles only to certain ends.
Example of this:
- Nerve cells –> exocytosis only occurs at the synapses.
- Epithelial cells –> the sides can be fused through tight seals in cell adhesion –> forces the movement of the content from the bottom of the cell upwards.
What is endocytosis?
Endocytosis is a cellular process in which substances are brought into the cell.
What are the three main types of endocytosis?
- Pinocytosis –> small molecules suspended in a fluid are brought into the cell (drinking).
- Phagocytosis –> cell eating and taking in larger material. E.g. recycling of material/ removal of apoptotic cells
- Receptor-mediated –> ligand has bound to specific receptor and cell brings it in to pass on signal or down regulate signal.
Difference between micro and macro endocytosis?
Endocytosis which is on a small scale with small vesicles –> micro
Endocytosis which is on a large scale with larger vesicles –> macro
Macro is present in ‘hungry’ cells which could be a sign of cancer.
How does cell know what type of endocytosis to use?
Different cell types will use these types in different ways –> This may change depending on whether the cell is dividing, starving or becomes cancerous.
Role of endocytosis in the immune system?
Important for immune system –> method used for surveillance of surroundings –> what is happening to the surrounding tissues –> if pathogen present –> macrophages can engulf them using phagocytosis.
However, Pathogens have also evolved ways to use endocytosis to enter the cells.
What are the characteristics of pinocytosis?
- Fluids and solutes are taken up
- Small vesicles about 100nm
- Most eukaryotic cells do it continuously without signals —> continuous monitoring of environment or boosting nutrient uptake (epithelial cells) –> This is constitutively
- Can remove damaged membrane?
Outline the process of vesicle formation –> i.e. when a vesicle is formed during endocytosis.
Two key proteins –> Clathrin and Caveolae
- Clathrin forms coats and allows the vesicles to form and take in extracellular fluid –> once vesicle is formed –> coat is shed to form a naked vesicle.
- Caveolae don’t form coats –> They are made up of caveolins and cavin proteins and are present in the PM of most cells, mainly in lipid rafts –> Responsible for PM bending into flask like shapes –> Caveolae are not shed.
- Lastly –> Dynamin wraps around the neck of the vesicle and pinches it off
- After this the vesicle can fuse with endosomes –> Endosomes can be classified into early/ late/ recyclable depending on how long they have been present in the cell and the markers present.
Definition of Transcytosed?
Transcytosed = taken right across the cell –> E.g. the epithelial cells in the gut, inside to the circulatory system.
Apart from vesicle formation what other role do caveolae play?
Cells use caveolae to know if they are being stretched or squashed
For example:
- Caveolin helps the cell know if it is being stretched –> Cavin proteins popped off –> to signal the inside of the cell –> response could increase cytoskeleton around the area to resist changes
Why is it difficult to prevent viruses from entering into the cell?
What are the characteristics of phagocytosis?
Phagocytosis
- Taking up larger material + some fluid will enter
E.g. microorganisms and dead cells
- Contents are broken down to make it safe for the cell and recycle
- Size >250nm phagosomes
Outline what ‘professional phagocytes’ (macrophages and neutrophils) do with the phagosome once it enters the cell.
Macrophages have a role in wound healing and regeneration response
- Contents are taken in via endocytosis
- Phagosome fuses with a lysosome
- Enzymes within the lysosome to break down the material.
- Permeases will allow molecules to be reused in the cytoplasm.
- Residual R bodies will be removed via exocytosis
Outline the characteristics of receptor-mediated endocytosis.
- Involves a ligand binding to a receptor in order to trigger endocytosis
- Receptors are concentrated within a particular area –> Sometimes this has already occurred in a lipid raft.
- This type of endocytosis is used when specific molecules/substances are required from circulation.
- All receptors to undergo this process use clathrin
- Sometimes a mix of cargo can be present in a single vesicle.
Explain step by step the process of receptor-mediated endocytosis using LDL cholesterol as an example.
LDL cholesterol –> cholesterol associated with a protein –> so that it can be transported through the blood.
- Protein portion recognised by LDL receptors
- Adaptin binds to the inside of the receptors
- Adaptin recruits clathrin which coats the membrane
- Bending of membrane and formation of vesicle
- Inside the cell it uncoats and fuses with endosome
- Low internal pH causes LDL receptor to release cargo
- Returned in a vesicle to the PM?
- Cycle occurs every 10 mins
- Protein in LDL cholesterol Delivered to lysosome which has proteases to digest protein portion
- Cholesterol is then released into the cytosol for membrane synthesis.
Extra:
Some cells cannot take up cholesterol in disorders
Why? no/low receptors, other steps could not work
Result = high blood cholesterol, risk of coronary artery disease
How is iron taken up into cells?
Receptor mediate endocytosis - Iron uptake
- Transferrin and iron bind to a receptor on the cell
- Fuses with endosome
- Low pH releases iron
What are peroxisomes? General characteristics?
Peroxisomes are small, membrane-enclosed organelles that contain enzymes involved in a variety of metabolic reactions, including several aspects of energy metabolism.
Characteristics:
- Single membrane-bound
- Do not contain DNA or Ribosomes
- All their proteins are encoded in the nucleus
- These proteins are obtained by selective import from the cytosol. Some enter the membrane via the ER
- Contain oxidative enzymes e.g. catalase and urate oxidase –> Super high concentrations
- Major sites of oxygen utilisation
- They are self-replicating
Outline how Peroxisomes use molecular oxygen and hydrogen peroxide to perform oxidation reactions.
- Use molecular oxygen to remove hydrogen atoms from organic substrates –> in an oxidation reaction producing H2O2
RH2 + O2 –> R + H2O2
- Catalase uses the hydrogen peroxide generated by the other enzymes to generate other substrates
E.g. formic acid, formaldehyde and alcohols
This is through the peroxidation reaction H2O2 + R’H2 –> RI + 2H2O
- Hydrogen peroxide is broken by catalase: 2H2O2 –> O2+2H2O –> bad for cells as it forms reactive oxygen species.
Examples of peroxisome action?
- Key in liver and kidney cells to detoxify harmful molecules present in the blood
E.g. ethanol –> acetaldehyde
- A major function in the breakdown of fatty acids to acetyl CoA which is called β oxidation, by blocking off 2x carbon atoms at a time –> used for the citric acid cycle.
- Catalyse formation of plasmalogens = phospholipids in myelin –> Plasmalogen deficiencies from peroxisomal disorders lead directly to neurological diseases
How are proteins imported into peroxisomes?
- Ser-Lys-Leu located at c terminal functions as the import signal –> If the sequence is attached to cytosolic proteins it will be imported into the peroxisome after being recognised by soluble receptors in the cytosol.
- Transport involves –> Peroxin proteins + ATP hydrolysis-driven process
- Six different peroxins form a protein translocator at the peroxisome membrane where unfolding of proteins does not need to occur –> The pore is dynamic, adapting in size for each cargo.
- Pex5 accompanies cargo into the peroxisome before returning to the cytosol –> The return is due to the removal of ubiquitin
What does a Mutation in Pex5 causes? (peroxisomes)
Mutation in Pex5 causes severe deficiency which leads to kidney and liver problems and death soon after birth (Zellweger syndrome).
Where are peroxisome membranes formed?
- Most peroxisome membranes are made in the cytosol then inserted into existing peroxisomes
- Others are made in the ER as precursor vesicles (also a way of bringing in proteins to existing peroxisomes where they fuse with others and take in proteins to become mature peroxisomes)
Outline some major process that peroxisomes are involved in.
Involved in:
- Penicillin biosynthesis in fungi
- Seed lipids to carbohydrate
- Carbon recovery in photosynthesis
- Cholesterol synthesis (HMG-CoA reductase, is ‘statin’ target)
- Bile acid synthesis (from cholesterol in liver)
- Synthesis of plasmalogens (fatty acid + glycerol with ether bond, heart and brain)
- Breakdown of excess purines (AMP, GMP) to uric acid
- Degrade several xenobiotics (a substance found within an organism that is not naturally produced or expected to be present within the organism)
- Break down eicosanoids (signalling molecules) (can modulate inflammation)
What is the extracellular matrix?
The extracellular matrix (ECM) is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins, that provide structural and biochemical support of surrounding cells.
What is the basal lamina? General characteristics?
The basal lamina is a layer of extracellular matrix secreted by the epithelial cells, on which the epithelium sits.
- Varied in composition depending on the need of the cell. E.g. wraps around skeletal muscle
- Thin 40-120nm
- Tough and flexible
- Under the epithelia and around muscle
- Filtering properties e.g. in the kidneys
- Promotes cell survival (source of stay alive signals), division, differentiation etc.
What is the basal lamin made of?
1) proteoglycans, highly negatively charged e.g. GAGs
2) fibrous proteins, mainly being collagen
3) non-collagen proteins
What are GAG proteins used for in the extracellular matrix?
- GAGs attract water and swell to occupy lots of space with low mass.
- Useful for hollow tube formation or gels to absorb forces
- Common ones: aggrecan, dally and betaglycan
What are fibrous proteins useful for in the extracellular matrix?
- Fibrous proteins: usually collagens –> resist tensile forces.
- 42+types in humans
- Assemble into fibrils then fibres –> Collagen spontaneously self assembles into a fibril
- Lack of any of these can lead to diseases e.g. fragile skin that cannot resist pulling forces so tears and blisters –> E.g. fibril forming, network forming, fibril-associated
What are non-collagen glycoproteins useful for? (Lamins and integrins)
Laminins
- 3 chains that form coiled coils with disulphide bridges –> Self-assembly into mesh held into place by integrins and basal lamina receptors
- Cell can secrete as much is required, under stress, It will secrete more to create a stronger mesh
- Mutations in them have predictable results.
- If it is a cancer cell it can secrete enzymes to break it down and metastasize
Integrins –> Matrix receptors
- Allow for communication between the extracellular matrix and the cell (bidirectional communication) –> connects ECM with cell –> they can be altered in order to elicit a response that changes the cell or the matrix.
- They are Allosteric regulated
- No ligand bound –> folded up and collapsed
- When the ligand (talin) is introduced –> conformational changes of the inside and the outside of the cell allows it to straighten up and convey signals –> activates integrin
Which two broad examples can be used to explain cell adhesion and the ECM?
- Connective tissue (bone or tendon) –> are formed from an extracellular matrix produced by cells that are distributed sparsely in the matrix –> It is the matrix rather than the cells that bear most of the mechanical stress to which the tissue is subjected –> direct attachment of cells is rare.
- The lining of the gut or the epidermal covering of the skin, cells are tightly bound together into sheets called epithelia –> ECM is less pronounced –> consisting mainly of a thin mat called the basal lamina –> in epithelium cells are connected by cell-cell junctions.
Outline the main cell-cell and cell-matrix junctions of epithelial cells.
Epithelial cells –> single layer of tall cells stands on a basal lamina (bottom) with the cells’ uppermost surface, or apex, in contact with the extracellular medium.
Laterally (between cells) –> cells formed junctions –>
- Two types of anchoring junctions link the cytoskeletons of adjacent cells.
1. Adherens junctions are anchorage sites for actin filaments
2. Desmosomes are anchorage sites for intermediate filaments - Two additional types of anchoring junctions link the cytoskeleton of the epithelial cells to the basal lamina
1. Actin-linked cell-matrix junctions anchor actin filaments to the matrix.
2. Hemidesmosomes anchor intermediate filaments to it.
What are tight junctions?
Tight junctions hold the cells closely together near the apex, sealing the gap between the cells and thereby preventing molecules from leaking across the epithelium.
What are Gap junctions
Gap junctions –> Near the basal end of the cells –> you have gap junctions that create passageways linking the cytoplasms of adjacent cells.
What are the two superfamilies that the transmembrane adhesion proteins for anchoring junction fall into?
- Cadherin superfamily –> mediate attachment of cell to cell
- Integrin superfamily chiefly mediate attachment of cells to matrix.
Specialization within each family:
For example some cadherins link to actin and form adherens junctions, while others link to intermediate filaments and form desmosomes.
Likewise, some integrins link to actin and form actin- linked cell-matrix junctions, while others link to intermediate filaments and form hemidesmosomes
NOTE –> Exceptions to these rules. Some integrins, for example, mediate cell–cell rather than cell–matrix attachment.
What is the most common and best understood cell-cell anchoring junctions?
Cadherins to link the cytoskeleton of one cell with that of its neighbour.
- Primary function is to resist the external forces that pull cells apart but at the same time it must be dynamic and adaptable, so that they can be altered or rearranged when tissues are remodeled or repaired,
What is homophilic adhesion?
Refers to the fact that –> Anchoring junctions between cells is symmetrical which is true in most cases.
For example:
- A linkage is to actin in the cell on one side of the junction, it will be to actin in the cell on the other side.
Likewise…
- Cadherin molecules of a specific subtype on one cell bind to cadherin molecules of the same or closely related subtype on adjacent cells.
Outline the general structure/mechanism of a cadherin molecule (extracellular region)
- All members of superfamily have an extracellular portion consisting of several copies of the extracellular cadherin (EC) domain (Classical –> 5 domains) –> Homophilic binding occurs at the N-terminal tips of the cadherin molecules (domains that lie furthest from the membrane) –> Terminal domain consists of a Knob and a pocket –> another cadherin (opposite cell) binds by inserting its knob into the pocket (not their own).
- Each cadherin domain unit –> connected to the next domain by a hinge –> when Ca2+ binds to hinge –> prevent it from flexing –> makes whole structure rigid –> Ca2+ removed –> the hinges flex –> structure becomes floppy –> mean the whole conformation at the N-terminus is thought to change slightly –> weakening the binding affinity.
Are the cadherin to cadherin interaction strong?
No –> cadherins bind with low affinity to other cadherins.
But…
- Strong attachments result from the formation of many such weak bonds in parallel –> cadherin molecules are often clustered side-to-side with many other cadherin molecules on the same cell –>
Describe the role of Cadherin in organising developing tissue.
Cadherins form specific homophilic attachments –> Cadherins are not like glue –> Rather, they mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells. For example:
In vertebrate embryo –> changes in cadherin expression are seen in neural tube formation and pinching from the overlying ectoderm –> neural tube cells lose E-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to express E-cadherin.
Then when neural crest cells migrate from the neural tube –> cadherins become scarcely detectable and cadherin 7 appears that helps to hold migrating cells in groups –> cells then aggregate to form the ganglion –> overexpress N-Cadherin again.
How to cadherins bind to the cytoskeleton?
Intracellular domains –> interact with filaments of the cytoskeleton: actin at adherens junctions and intermediate filaments at desmosomes –> cytoskeletal linkage is crucial without it cadherins can’t stably hold cells together.
Linkage of cadherins to the cytoskeleton is indirect and depends on adaptor proteins that assemble on the cytoplasmic tail of the cadherin –> for example: at adherens junctions –> cadherin tails binds to Beta-catenin, p120-catenin, alpha-catenin (binds to beta-catenin and recruits other proteins to provide a dynamic linkage to actin)
The general structure of a classic cadherin?
- Made from 700-750 αα
- 5 extracellular cadherin domains
- Short cytoplasmic domain
- Proteins are glycosylated
- Strong cell-cell adhesion
- Links to cytoskeleton
What type of actin bundles are adherens junctions linked to? What is the consequence of this?
Most adherens junctions are linked to contractile bundles of actin filaments and non-muscle myosin II –> junctions are therefore subjected to pulling forces generated by the attached actin –> pulling forces are important for junction assembly and maintenance –> i.e. disruption of myosin activity results in the disassembly of many adherens junctions.
Furthermore, the contractile forces acting on a junction in one cell are balanced by contractile forces at the junction of the opposite cell –> doesn’t disrupt cells organisation in tissue –> junctions act as dynamic tension sensors that regulate their behaviour in response to changing mechanical conditions.
We do not understand the mechanisms responsible for maintaining this balance –> some behaviour is due to proteins in the cadherin complex altering shape –> α-catenin is stretched under tension –> exposes a cryptic binding site for another protein, vinculin, which promotes the recruitment of more actin to the junction
Outline the importance of Cell-Cell adhesion in tissue modelling.
Example using epithelial.
Indirectly linking the actin filaments in one cell to those in its neighbours –> enables the cells in the tissue to use their actin cytoskeletons in a coordinated way –> allows cells to work together in order to form a specific type of tissue.
Epithelial cells –> adheren junctions form an adhesion belt –> junction between cells consisting of cadherins –> present along all cell in epithelial layer –> in each cell a bundle of actin and myosin lies adjacent to the belt and is attached to adheren junctions via adaptor proteins –> forms an extensive transcellular network.
Application –> bundles of actin and myosin filaments running along adhesion belts causes the epithelial cells to narrow at their apex (contract) and helps the epithelial sheet to roll up into a tube. An example is the formation of the neural tube –> like forming a vesicle
What are desmosomes and what is their function?
Desmosomes are structurally similar to adherens junctions but contain specialized cadherins that link to intermediate filaments instead of actin filaments.
Their main function is to provide mechanical strength –> possible because bundles of ropelike intermediate filaments that are anchored to the desmosomes form a structural framework of great tensile strength
What other key cytoplasmic protein is important for tight junction formation?
Key organizational proteins at tight junctions are the zonula occludens (ZO) proteins (3 types) –> large scaffold proteins that provide structural support on which the tight junction is built.
These molecules –> consist of strings of protein-binding domains –> recognize and bind the C-terminal tails of specific partner proteins (Claudin protein, occludin, actin cytoskeleton or to other scaffold proteins) –> creates a ‘mat’ that organizes the strand within the tight junction.
Outline the structure of Connexins (gap junctions)
Connexins are four-pass transmembrane proteins, six of which assemble to form a hemichannel or connexon.
When connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors.
A junction is formed by many such connexon pairs in next to eachother, forming a sort of molecular sieve.
Can vesicles from the Golgi go directly to the cell surface to undergo exocytosis?
Yes –> vesicles can be transported directly to the cell surface for exocytosis.
Entry into this pathway does not require a particular signal, it is also called the default pathway.
What happens to vesicles once they pinch off the TGN?
Initially –> vesicles that pinch off from the TGN –> not very concentrated/protein is loosely wrapped by membrane –> immature vesicles –> but as the vesicles mature –> they fuse with one another and their contents become concentrated or the membrane is recycled (vesicles bud off back to Golgi –> clathrin-coated)
Outline the basic idea behind receptor-mediated endocytosis.
- The ligand/macromolecule binds to the receptor –> which are accumulated in the coated pits.
- Results in an actin rearrangements
- Enter the cell as receptor–macromolecule complexes in clathrin-coated vesicles
This process –> increases the efficiency of internalization of particular ligands more than a hundredfold.
How is LDL cholesterol transported into cells?
Cholesterol is found in the blood as cholesteryl esters in the form of lipid-protein particles known as low-density lipoproteins (LDLs).
What happens to the LDL receptor in the early endosome?
In the early endosome, the LDL receptor dissociates from its ligand, LDL, and is recycled back to the plasma membrane for reuse, leaving the discharged LDL to be carried to lysosomes
The recycling transport vesicles bud from long, narrow tubules that extend from the early endosomes.
What is the role of intraluminal vesicles in endosomes?How are they formed?
Intraluminal vesicles carry endocytosed membrane proteins that are to be degraded –> this way receptors and any signalling proteins strongly bound to them are sequestered away from the cytosol (like EGF receptors)
They are also are made fully accessible to the digestive enzymes that eventually will degrade them
Sorting into intraluminal vesicles requires one or multiple ubiquitin tags –> added to cystolic domains of proteins –> tags recognised by cytosolic ESCRT protein –> mediate formation of intraluminal vesicle + process requires lipid kinase phosphatidylinositol to produce PI(3)P, which serves as an additional docking site for the ESCRT complexes.
Both PI(3)P and the presence of ubiquitylated cargo proteins to membrane.
Outline how actin is used to form pseudopods.
Actin polymerization, initiated by Rho family GTPases and their activating Rho- GEFs allows for the formation of these cellular extensions (pseudopods).
Activated Rho GTPases switch on the kinase activity of local PI kinases to produce PI(4,5)P2 in the membrane, which stimulates actin polymerization.
Seal off the phagosome and complete the engulfment, actin is depolymerized by a PI 3-kinase that converts the PI(4,5)P2 to PI(3,4,5)P3, which is required for closure of the phagosome.
