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 are tight junctions and what are they used for?
Tight junction acts like a barrier between cells to prevent molecules from leaking freely across the cell sheet –> From Apical end to the basal end.
Outline the importance of tight junctions in the small intestine.
The transcellular transport (from gut into the blood) depends on two types of transport.
- Apical transport –> active transport of molecules into the cell
- Basolateral transport –> same molecules to leave the cell by passive transport into the extracellular fluid on the other side of the epithelium.
For this to be effective –> spaces between the epithelial cells must be tightly sealed, so that the transported molecules cannot leak back into the gut lumen through these spaces
Additional –> the tight junction functions to keep the membrane proteins required for transport on the apical membrane within that region (proteins diffuse within the membrane) (also applies to basolateral) –> help prevent apical or basolateral proteins from diffusing into the wrong region.
Describe the structure of the tight junctions.
Branching network of sealing strands that completely encircles the apical end of the cell.
- The sealing strands are composed of a long row of transmembrane homophilic adhesion proteins embedded in each of the two interacting plasma membranes –> domains of these proteins adhere directly to one another to occlude the intercellular space
- Main transmembrane proteins forming these strands are the claudins (structure and assembly) + Normal tight junctions also contain a second major transmembrane protein called occludin (limits permeability) –> third transmembrane protein, tricellulin, is required to seal cell membranes together and prevent transepithelial leakage
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.
What are gap junctions?
Gap junctions –> bridges gaps between adjacent cells so as to create direct channels from the cytoplasm of one to that of the other.
Pore allows the exchange of inorganic ions and other small water-soluble molecules, but not of macromolecules such as proteins or nucleic acids.
The gap is spanned by channel-forming proteins, of which there are two distinct families, called the connexins (vertebrates) and the innexins (invertebrates).
Example of gap junction within in heart cells.
Electrical coupling via gap junctions serves an obvious purpose in tissues containing electrically excitable cells action –> basically allows action potentials tp spread rapidly from cell to cell.
In vertebrates, for example, electrical coupling through gap junctions synchronizes the contractions of heart muscle cells
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.
How can gap-junctions differ from each other between cells?
Gap junction in different cells –> different properties –> different connexins joined together –> channels with different permeability and regulation.
Most cells express one type of connexin but it is possible to that two different connexin proteins can assemble into a heteromeric connexon.
Additionally –> as each cells contributes half a channel –> two aligned half- channels can be different
Do gap-junctions stay open all the time?
Individual gap-junction channels do not remain open all the time; instead, they flip between open and closed states.
Change triggered by different stimuli –> voltage, membrane potential, chemical properties of cytoplasm (pH, Ca2+ concentration, etc).
In plants, how do cells communicate with each other?
Plant cells have only one class of intercellular junctions, plasmodesmata.
Like gap junc- tions, they directly connect the cytoplasms of adjacent cells.
What are the three main major classes of macromolecules in the ECM?
(1) glycosaminoglycans (GAGs), which are large and highly charged polysaccharides that are usually covalently linked to protein in the form of proteoglycans;
(2) fibrous proteins, which are primarily members of the collagen family;
(3) a large class of non-collagen glycoproteins, which carry conventional asparagine-linked oligosaccharides.
All three classes of macromolecule have many members and come in a great variety of shapes and sizes.
Outline the general structure of GAGs.
Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units.
One of the two sugars in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated.
The second sugar is usually a uronic acid (glucuronic or iduronic)
Because there are sulfate or carboxyl groups on most of their sugars, GAGs are highly negatively charged
4 main types of GAGs?
(1) hyaluronan
(2) chondroitin sulfate and dermatan sulfate
(3) heparan sulfate
(4) keratan sulfate.
What conformation do GAGs adopt?
Polysaccharide chains are too stiff to fold into compact globular structures, and they are strongly hydrophilic.
Thus, GAGs tend to adopt highly extended conformations that occupy a huge volume relative to their mass and they form hydrated gels even at very low concentrations
Why are GAGs associated with a large amount of water? What is the consequence of this?
Their high density of negative charges attracts a cloud of cations, especially Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix. This creates a swelling pressure, or turgor, that enables the matrix to withstand compressive forces.
Cartilage matrix that lines the knee joint, for example, can support pressures of hundreds of atmospheres in this way
Characteristics of Hyaluronan (a type of GAG).
- Simplest of the GAGs
- Regular repeating sequence of up to 25,000 disaccharide units
- no sulfated sugars
- all its disaccharide units are identical
- Its chain length is enormous
- Generally not linked covalently to any core protein.
Except for hyaluronan, are all other GAGs linked to protein cores? If yes, how are the polysaccharides added to the protein?
Yes, all other GAGs are covalently attached to protein as proteoglycans, which are produced by most animal cells –> polysaccharide chains are mainly assembled on this core protein in the Golgi apparatus.
Tetrasaccharide is attached to a serine side chain on the core protein to serve as a primer for polysaccharide growth; then, one sugar at a time is added by specific glycosyltransferases –> in Golgi the polysaccharide is modified by enzymes sequentially.
How to distinguish glycoproteins for proteoglycans?
Proteoglycan –> at least one of the sugar side chain of a proteoglycan must be a GAG (repeating disaccharide units) –> larger percentage of carbohydrates –> up to 95% by weight.
Glycoproteins generally contain relatively short, branched oligosaccharide chains that contribute only a small fraction of their weight.
What is a major protein in the ECM in humans?
Collagen –> secreted in large quantities by connective-tissue cells, and in smaller quantities by many other cell types.
- As a major component of skin and bone, collagens are the most abundant proteins in mammals.
- The primary feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called α chains, are wound around one another in a ropelike superhelix.
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 do cells that are specialized for secretion do with their vesicles?
Cells that are specialized for secreting some of their products rapidly on demand concentrate and store these products in secretory vesicles.
Vesicle produced in TGN –> release content in extracellular space via exocytosis.
The secreted product can be either a small molecule (such as histamine or a neuropeptide) or a protein (such as a hormone or digestive enzyme).
What is the process called that is used to package proteins into secretory vesicles?
Proteins destined for secretory vesicles (called secretory proteins) are packaged into appropriate vesicles in the TGN by a mechanism that involves the selective aggregation of the secretory proteins.
Proteins that are destined to be exported containing specific sorting signals –> tells the TGN to put it into a secretory vesicle.
However, It is unclear how the aggregates of secretory proteins are segregated into secretory vesicles.
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)
Apart from increasing concentration, what else occurs in a maturing vesicle?
Formation of active/mature proteins.
Protein hormones, small neuropeptides, secreted hydrolytic enzymes, are synthesized as inactive precursors –> Proteolysis is necessary to liberate the active molecules from these precursor proteins –> These cleavages occur in the secretory vesicles and sometimes in the extracellular fluid after secretion.
Plus, many of the precursor proteins have an N-terminal pro-peptide that is cleaved off to yield the mature protein.
Why is proteolytic processing so common in the secretory pathway?
- Some peptides are simply too short to be cotranslationally transported into the ER lumen (enkephalins).
- Hydrolytic enzymes or any other protein whose activity could be harmful inside the cell –> delaying activation is crucial
Once a secretory vesicle is loaded what happens?
Once loaded, a secretory vesicle has to reach the site of secretion, which in some cells is far away from the TGN –> can be quite long –> Nerve cells –> peptide neurotransmitters have to travel from cell body to axon.
Transport vesicles containing materials for constitutive release fuse with the plasma membrane once they arrive there –> secretory vesicles in the regulated pathway wait at the membrane until the cell receives a signal to secrete (action potential or extracellular signal –> any case leads to Ca2+ concentration increase in the cytosol).
What are the two types of vesicles stored by neurons?
- Vesicles released by the regulated secretory pathway.
- Specialized secretory vesicle –> synaptic vesicles
What are synaptic vesicles?
Synaptic vesicles store small neurotransmitter molecules, such as acetylcholine, glutamate, glycine, and γ-aminobutyric acid (GABA), which mediate rapid signalling from nerve cell to its target cell at chemical synapses.
Action potential arrives at a nerve terminal, it causes an influx of Ca2+ through voltage-gated Ca2+ channels, which triggers the synaptic vesicles to fuse with the plasma membrane
How are synaptic vesicles primed?
After vesicles have been docked at the presynaptic plasma membrane, they undergo a priming step, which prepares them for rapid fusion. In the primed state, the SNAREs are partly paired, their helices are not fully wound into the final four-helix bundle required for fusion.
Proteins called complexins freeze the SNARE complexes in this metastable state. The brake imposed by the complexins is released by another synaptic vesicle protein, synaptotagmin, which contains Ca2+-binding domains.
When Ca2+ binds –> Results in the displacement of the complexins.
How does the plasma membrane avoid constant increases in the surface area if exocytosis occurs?
Membrane components are removed from the surface by endocytosis almost as fast as they are added by exocytosis. Known as the endocytic–exocytic cycle.
What happens to the vesicles proteins that have been added to the membrane?
Vesicle proteins are either recycled or shuttled to lysosomes for degradation.
How can exocytosis be used to increase the membrane surface area?
An important task of regulated exocytosis is to deliver more membrane to enlarge the surface area of a cell’s plasma membrane
Outline the process of endocytosis?
In endocytosis, the material to be ingested is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form an endocytic vesicle containing the ingested substance or particle.
- Most eukaryotic cells constantly form endocytic vesicles, a process called pinocytosis.
- Some specialized cells contain dedicated pathways that take up large particles on demand, a process called phagocytosis
What happens with most endocytotic vesicle once it enters the cell.
Once generated at the plasma membrane, most endocytic vesicles fuse with a common receiving compartment, the early endosome.
Early endosome –> internalized cargo is sorted –> some cargo molecules are returned to the plasma membrane, either directly or via a recycling endosome, and others are designated for degradation by inclusion in a late endosome.
Are endocytotic vesicles Clathrin-coated?
Most vesicles formed during the process of endocytosis are clathrin-coated.
However… some vesicles may be formed using caveolae
Components of a caveolae vesicle.
Form lipid rafts in the membrane –> major structural protein in caveolae are caveolins -> proteins that insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane –> they are bound to large protein complexes of caving proteins in the cytoplasm –> stabilize membrane curvature.
Two examples of pathogens that use caveolae to enter the cell.
- Animal viruses such as SV40 and papillomavirus (which causes warts) enter cells in vesicles derived from caveolae –> delivered to early endosomes –> vesicle to lumen –> viral genome exits across the ER membrane –> imported into the nucleus to start the infection cycle.
- Cholera toxin –> also enters the cell through caveolae and is transported to the ER before entering the cytosol.
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).
Explain what happens when a cell needs to increases its uptake of LDL cholesterol from the blood.
Cell needs cholesterol for membrane synthesis –> makes receptor proteins for LDL and inserts into P.M –> once in the membrane LDL receptors diffuse until they associate with clathrin-coated pits –> Receptor binds to PI(4,5)P2 on the plasma membrane which unlocks conformation locally –> endocytosis signal in the cytoplasmic tail (LDL receptor) can now bind to the membrane-bound adaptor protein AP2 –> AP2 then recruits clathrin to initiate endocytosis.
Outline the process of receptor-mediated endocytosis of LDL cholesterol.
- LDL cholesterol binds to receptor –> taken in quickly into the cell.
- After shedding their clathrin coats, the vesicles deliver their contents to early endosomes.
- LDL and LDL receptors encounter the low pH in early endosomes –> LDL is released from its receptor.
- Delivered to late endosomes –> then to lysosomes
- In lysosome –> cholesteryl esters in the LDL particles are hydrolyzed to free cholesterol –> now available to the cell for new membrane synthesis.
What happens if a cell has too much cholesterol?
If too much free cholesterol accumulates in a cell, the cell shuts off both its own cholesterol synthesis and the synthesis of LDL receptors, so that it ceases both to make or to take up cholesterol.
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.
Outline the intake of transferrin receptors.
Transferrin is a soluble protein that carries iron in the blood.
- Receptor-mediated endocytosis of transferrin receptor + transferrin.
- Transferrin receptors + transferrin with its bound iron taken to early endosomes.
- The low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor
- Receptor–apotransferrin complex enters the tubular extensions of the early endosome and from there is recycled back to the plasma membrane.
- Apotransferrin returns to the neutral pH of the extracellular fluid, it dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again.
6.
What other pathway can receptors take (receptor-mediated endocytosis), apart from being recycled?
Use EGF as an example
Degradation by lysosomes
EGF is a small, extracellular signal protein that stimulates epidermal and various other cells to divide.
EGF receptors accumulate in clathrin-coated pits only after binding their ligand. (Ligand binds –> activates intracellular signal –> accumulation of complex in pits –> allows for it to be taken in and degraded)
Receptors degraded in lysosomes, along with the ingested EGF.
How are receptors downregulated by Degradation in Lysosomes?
Receptor downregulation is highly regulated.
- Activated receptors are first covalently modified on the cytosolic face with the small protein ubiquitin.
- Ubiquitin-binding proteins recognize the attached ubiquitin and help direct the modified receptors into clathrin-coated pits.
- In early endosome –> other ubiquitin-binding proteins –> recognise and sort the receptor into intraluminal vesicles –> ends up in late endosome
- The process is important as it prevents recycling of the receptor.
Characteristics of early endosomes?
- How are early endosomes created –> unclear but their membrane and volume are mainly derived from incoming endocytic vesicles that fuse with one another.
- Small and patrol the cytoplasm underlying the plasma membrane.
- Early endosomes have tubular and vacuolar domains –> most membrane surface in tubules and most of the volume in vacuolar domains.
- During maturation –> these domains have different fates –> Vacuolar retained and transformed into late endosome —> tubular region shrink (recycling) –> maturing endosomes migrate to the cell interior –> shedding vesicles which return to recycle to P.M or TGN + receive lysosomal proteins –> eventually vesicles fuse with each other as well with endolysosomes and lysosomes.
Outline the changes that occur during the maturation of endosomes.
- The endosome changes shape and location –> tubular lost + vacuolar shape modified
- Rab proteins, phosphoinositide lipids, fusion machinery (SNAREs and tethers), and microtubule motor proteins –> contribute to the makeover of cytosolic face of the membrane.
- V-type ATPase in the endosome membrane pumps H+ –> acidifies –> renders lysosomal hydrolases increasingly more active –> influences receptor-ligand binding.
- Intralumenal vesicles sequester endocytosed signalling receptor –> halting the receptor signalling activity.
- Lysosome proteins are delivered from the TGN to the maturing endosome.
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 endosomes can be useful for the process of transcytosis?
Example –> New-born obtaining antibodies from its mother’s milk
- Baby drinks mothers milk –> when in the lumen of the gut –> antibodies bind to the receptor (possible due to low pH)
- Receptor–antibody complexes are internalized via clathrin-coated pits vesicles and are delivered to early endosomes.
- Receptor Complex remains intact and is put on to a vesicle which is delivered to the basolateral membrane
- On exposure to the neutral pH of the extracellular fluid on the basolateral side –> antibodies dissociate from their receptors and eventually enter the baby’s bloodstream.
What is phagocytosis?
Phagocytosis is a special form of endocytosis in which a cell uses large endocytic vesicles called phagosomes to ingest large particles such as microorganisms and dead cells.
Few cells in multicellular organisms are able to ingest such large particles efficiently –> carried out mainly by specialized cells—so-called professional phagocytes.
What are the two classes of white blood cells that act as professional phagocytes?
Two important classes of white blood cells that act as professional phagocytes are macrophages and neutrophils –> They ingest invading microorganisms to defend us against infection and also scavenging old cells and cells that have died by apoptosis
What happens with a phagosome once it enters the cell?
Phagosomes fuse with lysosomes and the ingested material is then degraded –> Indigestible substances remain in the lysosomes, forming residual bodies that can be excreted from cells by exocytosis.
Example of pathogenic bacteria that have developed mechanisms to prevent phagosome-lysosome fusion?
The bacterium Legionella pneumophila —> injects into its host a Rab-modifying enzyme that causes certain Rab proteins to misdirect membrane traffic, thereby preventing phagosome-lysosome fusion.
Consequently –> bacteria remains in the modified phagosome, growing and dividing as an intracellular pathogen, protected from the host’s adaptive immune system.
How is phagocytosis triggered?
Phagocytosis is a cargo-triggered process –> requires the activation of cell-surface receptors that transmit signals to the cell interior.
Particles must first bind to the surface of the phagocyte –> phagocytes have a variety of receptors –> that are functionally linked to the phagocytic machinery.
Outline how antibodies are used for phagocytosis.
- Antibodies initially attack a pathogen –> they coat it with antibody molecules
- Antibody molecules can bind to Fc receptors on the surface of macrophages and neutrophils.
- Activates the receptors to induce the phagocytic cell to extend pseudopods, which engulf the particle and fuse at their tips to form a phagosome
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.
Why do phagosomes not ingest cells of their own body?
The living cells display “don’t-eat-me” signals in the form of cell-surface proteins that bind to inhibiting receptors on the surface of macrophages.
Inhibitory receptors recruit tyrosine phosphatases that antagonize the intracellular signalling events required to initiate phagocytosis.
Like many other cell processes, depends on a balance between positive signals that activate the process and negative signals that inhibit it.
