Lecture Notes on Cell Biology PDF

Lecture 4 (2) Edition 2 A good example is the sperm cell. Although it is a free roaming cell, yet it exhibits a domain specific distribution of the proteins. And thus certain limitations of distribution are still not clear. Phospholipid Composition: Different regions of the sperm membr...

Lecture 4 (2) Edition 2 A good example is the sperm cell. Although it is a free roaming cell, yet it exhibits a domain specific distribution of the proteins. And thus certain limitations of distribution are still not clear. Phospholipid Composition: Different regions of the sperm membrane have distinct lipid compositions, which can recruit and stabilize specific proteins. For example, lipid rafts in the tail region can concentrate proteins involved in motility. Attachment to the Cytoskeleton: The cytoskeleton plays a crucial role in maintaining the position of proteins. In sperm cells, the cytoskeletal elements like actin and microtubules help anchor proteins in specific domains Biol 260 Lecture 5 (1) ED.2 I. Introduction: The lipid bilayer is impermeable to water-soluble molecules therefore special ways are needed to transfer water-soluble molecules into cells (i.e. across membrane). So not all molecules can easily cross the membrane and cells must transport many types of molecules across the membrane, and ALL cells are capable of transporting molecule in/out across. So cells can: 1. Ingest nutrients 2. Excrete waste 3. Regulate intracellular ion conc. 4. Transport small water-soluble molecules into cell (like glucose) 5. Transport of large water-soluble molecules into/out of cell (ex. proteins, enzymes). Into by endocytosis and out by exocytosis (like enzymes, ECM proteins...). The cell has developed methods to achieve all these types of transport. NOTE: It is evident that selective permeability is in effect. Therefore, large differences exist in the ionic composition of cytosol relative to outside environment. This difference is a potential energy that is used to drive various transport processes across the membrane. II. Generic permeability properties of the plasma membrane (Lipid bilayers in general): A very important rule to remember is that the smaller(hence fewer H bonds with H2O) and the more hydrophobic the molecule is the easier and the faster for it to cross the lipid bilayer. The relative ease with which a molecule passes through depends on these factors as well. So from the figure we can deduce that we have 4 main types of molecules crossing the lipid bilayer: - Small hydrophobic molecules: like O2 and CO2, steroid hormones, can diffuse easily. Some even have channels. - Small uncharged polar molecules: like water, urea and glycerol. These can cross the membrane by diffusion (but it is quite hard). Water gets in & out by channels: Aquaporins(their discoverer won the noble prize for medicine). - Large uncharged polar molecules: like glucose and sucrose. These cannot get in by diffusion across the membrane. Channels or carrier proteins are required. - Ions:Na+, K+...etc. Ions cannot cross the membrane, so carriers or channels are needed. Note: The relative ease with which this or that molecule crosses a LIPID BILAYER is dependent on these factors. Biol 260 Lecture 5 (1) ED.2 For example water is times more permeable than sodium or potassium ions, meaning that for each passing 10 billion water molecules 1 sodium ion passes, and this is nearly null, so practically no Na+ diffuses across the bilayer (NOTE:Figure 11.2:these numbers are in a lipid bilayer in a black membrane or liposome with no carriers or channels since we cannot get such membrane proteins to function in such models). III.Transport of small water soluble molecules across the membrane: To transport small water soluble molecules across the membrane you need transport proteins: CHANNELS VS CARRIER PROTEINS Some important properties: - They are present in ALL types of cells - They exist in different types and subtypes - They can be present on the apical or basal parts of the membrane(But not on lateral side) - They (the different types and subtypes) have a tissue specific distribution, where the transport proteins types present in a neuronal cell are different from those in an intestinal cell and so on (and this can be said for the ECM, BM and cytoskeleton which are also specific to their tissue) The differences between the two proteins: CHANNELS: - Are continuous protein pathways across the lipid bilayer - Undergo no conformational change to allow the passage of the molecule across the membrane - Allows the passage of only one type of molecule through it - Allows more amounts of molecule to pass and in a faster manner than carrier proteins - Only passive transport occurs through channels Biol 260 Lecture 5 (1) ED.2 CARRIER PROTEINS: - To transport the solute across the membrane it must undergo a conformational change: Bind solutes on one side of membrane /Undergo a conformational change/Thus, expose solute to other side of membrane - Can transport one type of molecule (Uniport), or two or more types of molecules can be transported by the same carrier (Coupled), for the coupled the carrier can be a symport(molecules transported in the SAME direction) or antiport(molecules transported in opposite directions). - Carriers transport less amounts of molecules and in a slower manner than channels. - They can be engaged either in activetransport (uphill) driven by a series of conformational changes via ATP hydrolyses or via ion binding, or in passive transport (downhill) where no energy is needed. NOTE: A single type of molecule can be transported by both channels and carriers. Ex: Na+ Biol 260 Lecture 5 (1) ED.2 Example of a carrier: Na+/K+ pump: 3 Na+ bind the carrier protein (a transmembrane one) conformational change due to Phosphorylation Na+ released Conformational change 2 K+ bind dephosphorylating 2K+ released to the other side of the membrane. So the Na+/K+ pump is an example of: - Carrier mediated transport - Active transport - Coupled transport - An antiport Biol 260 Lecture 5 (1) ED.2 Another example is the glucose pump where glucose is brought in with Na+ (active transport): A small recap:A cell will have carriers and channels of different types and these types will differ from one cell to the other and these are distributed on the apical and basal sides of the membrane. So in the figure above we notice that the cell has Na+/K+ pumps (antiport), Na+/glucose transporter (symport), glucose carriers (uniport) and many others. This is in addition to channels (for Na+, K+, Cl-) and these can all be present on a single of cell, but the number and their different types and subtypes will differ between tissues, the same goes for both channels and carriers.  Biol 260 Lecture 5 (1) ED.2 More info about channels: Form aqueous pores across bilayer and hence allow inorganic ions to cross membrane downhill (solutes move across at least 100 times faster than their transfer by carriers). Channels can be open or closed and they come in several types: - Voltage-gated(electric potential gated)(open upon a threshold of potential difference across the membrane(change in membrane potential) ex: in neurons and muscle cells) - Ligand or chemically gated (gates open upon binding to a molecule, like a neurotransmitter) - Mechanically gated (a protein pushes it to open) Example on the complexity of membrane transport proteins: The synapse during muscle contraction: During muscle contraction, action potential arrive to the presynaptic cell of a neuromuscular junction and cause the movement of the Ach containing vesicles in the membrane, the vesicles roll down the cytoskeleton through the activation of motor protein. The vesicle will fuse with the membrane and release the neurotransmitters by exocytosis. The neurotransmitter now in the synaptic cleft diffuses towards its receptor (which is a chemically gated ion channel) by affinity.(note: the receptor is made up of 5 transmembrane proteins that come together to form two pockets that are complementary to the neurotransmitter). Action Potential Arrival: An action potential (electrical signal) arrives at the presynaptic terminal of a motor neuron at the neuromuscular junction. Release of Acetylcholine (ACh): This action potential triggers the movement of vesicles containing the neurotransmitter acetylcholine (ACh) towards the presynaptic membrane. These vesicles move along the cytoskeleton with the help of motor proteins. Exocytosis: The vesicles fuse with the presynaptic membrane and release ACh into the synaptic cleft through a process called exocytosis. Binding to Receptors: ACh diffuses across the synaptic cleft and binds to its receptors on the postsynaptic membrane of the muscle cell. These receptors are chemically gated ion channels made up of five transmembrane proteins that form two binding pockets for ACh.  Biol 260 Lecture 5 (1) ED.2 So upon binding, the channel opens, and (see the figure): Na+ rushes in=> change in membrane potential=>opens voltage gated Na+ channels=>more Na+ rushes in=>Proteins push to open mechanically gated Ca++ channels=>Ca++ rushes to cytoplasm=>Ca++ sits on troponin C, which is part of the troponin/tropomysin complex => troponin C moves away=>Causing it to release myosin and actin=>Actin and myosin interact and bind and slide which leads to muscle contraction. Opening of Ion Channels: Upon binding of ACh, these ion channels open, allowing Na+ ions to rush into the muscle cell. This influx of Na+ changes the membrane potential. Propagation of Action Potential: The change in membrane potential opens voltage-gated Na+ channels, leading to further influx of Na+ and propagation of the action potential along the muscle cell membrane. Opening of Ca++ Channels: The action potential travels along the muscle cell membrane and into the T-tubules, triggering the opening of mechanically gated Ca++ channels in the sarcoplasmic reticulum. Release of Ca++: Ca++ ions are released into the cytoplasm of the muscle cell. Binding to Troponin C: Ca++ binds to troponin C, a component of the troponin-tropomyosin complex on the actin filaments. So all this occurs in a split of a second, but a great deal of complexity is present in the activation of the channels and their interactions, and the involvement of many proteins. In addition, we notice that on the muscle specific types of receptors exist in this particular type of tissue in order to receive Ach and be activated by it. So we have shown how channels and carriers are very complex and distributed in a tissue specific manner. Conformational Change: This binding causes a conformational change in troponin C, moving the tropomyosin away from the myosin-binding sites on actin. Cross-Bridge Formation: With the myosin-binding sites exposed, myosin heads bind to actin, forming cross-bridges. Muscle Contraction: The myosin heads pivot, pulling the actin filaments towards the center of the sarcomere. This sliding of actin over myosin results in muscle contraction. Relaxation: When the action potential ceases, Ca++ is pumped back into the sarcoplasmic reticulum, troponin C returns to its original shape, tropomyosin covers the myosin-binding sites on actin, and the muscle relaxes. Biol 260 Lecture 5 (1) ED.2 IV. Transport of large water-soluble molecules across the membrane: We are not talking here about small molecule like glucose or Na+ or K+. We are talking about large water-soluble molecules like PROTEINS. Because they are so large they go in and out of every single cell by Endocytosis and Exocytosis respectively. Exocytosis(More explanation in Lecture 6): A protein that is to be exocytosed means that it will be either secreted or become a membrane protein. Such proteins follow this pathway: mRNA protein ER Golgi secretion, or membrane. Proteins to be exocytosed must: - Decide whether to be released APICALLY or BASALLY. - They must go to the right vesicle that will take it to its right destination. - The protein can be released either in a constitutive(secreted when made)or regulated (made, retained and secreted at a later stage) manner. So a protein to be exocytosed is inserted/translocated into the ER. It will be glycosylated in the ER and then in the Golgi. Beyond the Golgi the proteins decides whether to go to the apical or basal side to be secreted and it decides to go their either in constitutive or regulated vesicle. This path is for all proteins to be SECRETED or Secretoryprotein (insulin, digestive enzymes, ECM proteins, secreted antibodies). For MEMBRANE Proteins, they travel in the same path as the secretory proteins but they go there by constitutive vesicles only. Constitutive vesicle: It is a vesicle that once the protein is in it, it takes the protein immediately to the membrane. Regulated vesicle:Takes up the protein then stays on hold in the cytosol until the cell receives a signal from outside telling it to move the vesicle to the membrane (most hormones are secreted by regulated vesicles). So all vesicular trafficking goes from ER=>Golgi=>Beyond. This is mediated by Coated Vesicles. ALL vesicles in the cell are coated with proteins of different types and shapes. Three major coat protein families in the cell are CLATHRIN proteins, COPI (coatmer protein I) or COPII. Biol 260 Lecture 5 (1) ED.2 So what is the coat for? The coat is essential for the vesicle in receiving the right cargo and taking it to the right t or a marker that guides the right cargo to get into it and take it to the right destination. Ex: Insulin in the ER goes to the phospholipids with the right coat and once there the vesicle will form and take it where it needs to go. Insulin is driven to the phospholipids with the right coat by AFFINITY.NOTE: In addition to the coat, glycosylation (of the protein) is also essential in guiding the protein to its final destination. Types of coats: Vesicles from ER to Golgi are COPII coated All vesicles going back to the ER are COPI coated All vesicles going from Golgi to lysosome are Clathrin coated. Biol 260 Lecture 5 (1) ED.2 Additional info: 1. Constitutive pathway operates in all cells. 2. Regulated pathway: mostly in cells that are specialized in secretion upon demand. (I.e. cells that secrete hormones, neurotransmitters, digestive enzymes, etc) 3. Secretion of regulated vesicle is stimulated by a hormonal signal or other similar signal. 4. Certain exocytic vesicles destined for secretion (or lysosome) are clathrincoate 5. NOTE: Other types of coats as COPI and COPII, coat transport vesicles in cytosol, however these will be discussed in details later. Biol 260 Lecture 5 (1) ED.2 Endocytosis 2 phenomena can be encompassed by this term: Pinocytosis - which involves the ingestion of fluid or solutes via small vesicles), and Phagocytosis - ingestion of large particles via large vesicles or phagosomes). -- Ingestion of microorganisms or cell debris (mainly done by specialized cells). But this is a misunderstanding and these two terms are meaningless. Pinocytosis can be used interchangeably with Endocytosis. NOTE: In contrast to previous beliefs all cells are capable of phagocytosis (not only immune cells), and this is because when a cell undergoes apoptosis it is phagocytosed by its neighboring cells. Figure 1: Electron micrograph of an endocytic vesicle So during endocytosis proteins bind to membrane receptors and start forming a pit that has: - The proteins or molecules to be endocytosed (the soluble ligand); these are non-steroid hormones, can be insulin, FSH (follicle stimulating hormone), ACTH, CKs (cytokines), growth factors, or just any hormone with a membrane receptor. - The coated pit, on the cytosolic side, made of clathrins - The transmembrane receptor The pit pinches inwards and forms a vesicle, this vesicle takes in with it physiological solution which contains water in addition to the soluble ligand=> so the misunderstanding was that this hat it gets in by other ways (aquaporins for example) and the term cellular drinking for pinocytosis is wrong. Important note about pinocytosis: Pinocytosis plays an essential role in signal transduction through three paths 1) Chemical signaling: When the ligand bind to the receptor, 2nd messengers are activated(like kinases), this would activate transcription factors that go into the nucleus and trigger gene expression 2) Physical signaling: The lateral movement of the coated pits causes a physical change in cytoskeleton then the nuclear matrix triggering signaling and gene expression. Biol 260 Lecture 5 (1) ED.2 3) Receptor down regulation:After endocytosis, by default some vesicles will go to the lysosomes for degradation, this will degrade the receptor and desensitizes the cell for coming ligands until it produces new receptors. Additional info: Unless specifically retrieved: Pinocytosis endosomes lysosomes (degraded via hydrolytic enzymes). Phagocytosis phagolysosomes lysosomes (degraded via hydrolytic enzymes). Lecture 5 (2) Summary of previous lecture - The transport of large water-soluble molecules across the membrane, either by exocytosis or endocytosis. - Exocytosis starts at the level of mRNA molecules, which are translated to proteins. Proteins then go to the ER and then to the Golgi. Then they are transported in a vesicle toward membrane for secretion or for positioning in the membrane. - Membrane proteins typically go to the membrane in a constitutive vesicle. - Regulated secretions. - Most vesicular trafficking is mediated by coated vesicles (have protein coats on them). There are various types of coats: Clathrin, COPI and COPII. The coat proteins help in the pinching off process of the vesicle and dictate which type of cargo goes into each vesicle. All cells secrete proteins; secretion is not specific to as liver cells, producing bile, and intestinal cells, producing hormones, secrete their products via the regulated pathway. (Note: most hormones are secreted via the regulated pathway.) Some non-secretory cells do not have the regulated pathway. All secretory cells have both the regulated and the constitutive pathways. Endocytosis (cont.) Brief reminder: - There is no such thing as cellular drinking or cellular eating. Pinocytosis and endocytosis are interchangeable terms. The term endocytosis is used is used more often - Endocytosis is an integral component of the signaling pathway. It is involved in mediating physical & chemical signaling and in the desensitization of the cell (down- regulation of receptors). - What are some of the distinctive features of endocytosis? 1. Clathrin is highly conserved, and it is ubiquitously expressed in all cells. With a few exceptions, the majority of endocytic vesicles are clathrin-coated. 2. Clathrin assemble into a network of hexagous or pentagous on the cytoplasmic side of the membrane. The invagination of the vesicle is driven by the assembled clathrin network (and their proteins within the network). Note: Clathrin molecules are made of three heavy chains and three light chains. Lecture 5 (2) 3. It associates with an ATPase known as Hsp70, and it associates with adaptins. Since clathrin is highly conserved, it is impossible that it binds to all the different endocytosed ligands unless all the ligands have the same consensus structure, which is not the case. The way clathrin solves this problem is through adaptins; adaptins are a family of proteins that come as pairs. Adaptins have a highly conserved side that binds to clathrin, and they have a highly variable side that binds to the various ligand-receptors (adaptins do not bind ligands directly). They are the bridge between clathrin molecules and ligands. Therefore, ligands bind to their receptors inducing a conformational change in the receptors, which leads to the recruitment and binding of adaptins. Adaptins then associate with the clathrin. As these complexes (ligand-adaptins-clathrin) undergo lateral diffusion in the membrane, clathrin starts to assemble at the coated pit initiating endocytosis and pinching off. The pinching off process is aided by the protein dynamin and other associated proteins. They grab the neck and help the endocytic vesicle pinch off. After the vesicle pinches off, the clathrins are shed off by ATP hydrolysis, (what the ATPase Hsp70 does). The shed clathrin is sent to cycling to the membrane to bring in more vesicles. Lecture 5 (2) Note: After pinching off of the vesicle, the membrane seals itself by the hydrophobic interactions (lateral diffusion of the phospholipids.) What are HSPs? HSPs are heat shock proteins initially discovered in bacteria. HSPs can sustain high temperatures (higher than 37 by 2-3 degrees). They were first discovered in bacteria: when bacteria were subjected to a heat shock, these proteins were expressed to engulf other proteins and keep them in shape (avoid denaturation). So HSPs in bacteria play a role similar to chaperones (not identical but similar). Nonetheless, proteins with sequence homology were later discovered in mammalian cells and hence called HSPs accordingly, but they do not have the same role as the bacterial HSPs. Some work as shedding clathrin off an endocytosed vesicle so that clathrin can re-circulate to the membrane Note: The function of chaperones differ than heat shock proteins in that (1) they help in the folding and not in preserving the folding and (2) they are not expressed only when there is an increase in temperature i.e. they are constitutively expressed. Some mammalian HSPs work as chaperones, and others have different functions, but none function as bacterial heat shock proteins. 4. The formation of endocytic vesicles in a cell is very frequent. There are thousands of events of endocytosis occurring at all times. 5. The default pathway for an endocytic vesicle is the lysosome. Unless the contents of the endocytic vesicle are specifically retrieved, Lecture 5 (2) 6. The lysosome is not a stable structure that awaits garbage to come to it as many think of it. The lysosome is always in the process of formation and degradation. It is always maturing and then it dies off. In the course of an endocytic event, the coated pit forms, then the shedding off of clathrin occurs, and then the formed endocytic vesicle and many other endocytic vesicles AND vesicles from the Golgi fuse together to form an early endosome, which matures into a lysosome. The hydrolytic enzymes in the lysosome come from the vesicles that bud off from the Golgi. In general, proteins go to the ER, then to the Golgi, and then vesicles either go towards the membrane or towards the lysosome to fuse with it. Vesicles heading to the lysosome have hydrolytic enzymes inside. So the endocytic vesicles fuse with vesicles coming from the Golgi that have the hydrolytic enzymes, and form the early endosome that matures temporally (with time) and spatially (as it moves) into a mature lysosome. How are early endosome and mature lysosomes different? The hydrolytic enzymes are initially in their inactive form. Later they are activated by the change in pH. When a vesicle pinches off from the Golgi (at pH 7), proton pumps in its membrane start working and pump protons to the inside decreasing the pH to almost 5.5 at which the hydrolytic enzymes become active in the mature lysosome. The reason why enzymes are not produced in the active form at first is to avoid the hydrolysis of needed cell components. Remember, the driving force for everything in the cell is affinity. 7. Different pathways of contents of endocytic vesicles; Ligand receptor complexes are sorted in the acidic environment of the endosome: Example 1: LDL-cholesterol pathway Legend Red = cholesterol Green = LDL Blue = LDL receptor Black= Hydrolytic enzymes Summary: LDL is endocytosed with its receptor. Clathrin is shed off. A naked vesicle forms the early endosome. The LDL receptor and the LDL are separated: the receptor is recycled back to the membrane whereas the LDL (with cholesterol) stays in the early Lecture 5 (2) endosome. The LDL is degraded by hydrolytic enzymes (as indicated by the absence green color seen in the lysosome), and hence freeing the cholesterol, which is retrieved to the cytoplasm to be used in other processes. What drives this process? Affinity drives the entire process. Numerical explanation of affinity: When two proteins are attracted to each other (such as antigen and Antibody, receptor and ligand, integrin about 10-12. This is the physiological Ka necessary for the assembly of any two such molecules (memorize it!). Ex. Antigen + Antibody Ag-Ab complex Ka = [complex]/[Ag][Ab] = 10-12 This means that the rate of complex formation is 1012 times more frequent than the rate of complex dissociation (high affinity). In other words, 10-12 means that when there are a trillion antibody- antigen complexes, only one complex would dissociate. The LDL receptor and the LDL have an affinity of 10-12. As they move into the cell, they lose this affinity because the pH changes. This continues, and the hydrolytic enzymes are capable enough to degrade the LDL, and the cholesterol is retrieved. The process of cholesterol retrieval was explained only two years ago whereas the process of endocytosis was in 1988 (to signify how long discoveries can take). So the change in pH causes this break-apart. But what causes the LDL-receptor to pinch off from the early endosome and head to the membrane? The steps of the process: (as Talhouk mentioned them) - Endocytosis - Shedding of clathrin - Drop in pH - Formation of early endosome - Separation of LDL from its receptor - LDL receptor travels to a specific location on the membrane for recycling and reuse - The LDL is degraded - The lysosome matures - The cholesterol is retrieved. The applications of this discovery in medicine are very important: In some people genetically prone to atherosclerosis, the LDL-receptor has a mutation. The receptor lacks the knob and cannot bind to adaptins and hence cholesterol cannot be internalized. Therefore, the cholesterol accumulates in the membranes of endothelial cells of blood vessels clogging them. (In coronary catheterization , physicians put oil and dye to check what is there. But the oil itself might wash the cholesterol out since they are both hydrophobic, so This is how coronary catheterization itself can be , it is detected by the dye.) Lecture 5 (2) epidermal growth factor- stimulates cell proliferation and growth and differentiation. Example 2: EGF-EGF receptor pathway: Both the ligand and the receptor are degraded in this case. Endocytosis is an integral part of signal transduction, and receptor down-regulation (desensitization) 1st path (chemical signaling): The EGF receptor is an RTK (Tyrosine Kinase receptor). When EGF binds to the receptor, there is lateral diffusion and endocytosis, and the receptor is activated. Since the receptor is an RTK (a kinase), it activates downstream signaling cascade that eventually leads to the phosphorylation of transcription factors that go into nucleus to trigger gene expression. 2nd path (physical signaling): The lateral movement affects the organization of the cytoskeleton, which affects the organization of the nuclear matrix, which in turn affects gene expression. 3rd path (desensitization): The vesicle is endocytosed and heads to the lysosome. Both the receptor and the ligand are degraded. So the cell becomes desensitized because of receptor down-regulation. If the cell still wants to be stimulated by EGF, it expresses new EGF receptors. etical pathway that is number Lecture 5 (2) EGF-EGF receptor path 1: Chemical Signal 2: Physical Signal 3: Down-regulation Example 3: Transferrin (next lecture) Example 4: IgD into milk (next lecture) Note: Endocytosis is not necessarily the outcome of ligand receptor interaction (from the outline). For example, some receptors may activate signaling pathways without being internalized BIOL 260 Lecture 5(3) Ed.1 Four Different pathways involved in endocytosis: Reminder: the default pathway of endocytosed particlesis endosome then lysosome, and lysosome is always in the process of formation(fusion of Golgi vesicle with endosome). First pathway: LDL (quickreview) The LDL molecules are degraded in lysosome and receptor is recycled back to membrane and cholesterol is absorbed in the cytosol. (This process was detected by Brown and Goldstein) Second pathway: EGF and EGF receptor. In the case of EGF, Both the ligand and receptor are degraded in the lysosome leading to receptor down regulation after activating the cascades involved in signal transduction. The mechanism of EGF activation involves four related processes: Physical pathway: the coated pit moves through the cytosol and alters its geometry, ultimately affecting gene expression Chemical pathway: the binding of EGF to its receptor activates RTKs leading to a cascade of signal transduction to activate TFs in the nucleus. Desensitization: both the ligand and receptor are degraded in the lysosome leading to receptor down regulation Debatable but possible: EGF molecule itself is cleaved in endosome and a part of it is retrieved in the cytosol. This part acts as a transcription factor itself and goes to nucleus to alter gene expression. Keep in mind: not all receptors are desensitized (some are recycled as in the case of LDL) Third pathway: Transferrin and Transferrin receptor (TF/TF-R) Transferrin (Tf) is an Iron-binding protein found in blood. It binds iron molecules in circulation, brings them to the cell, and then returns to circulation again. Notes: - during pregnancy. - Transferrin may help fight against Fe-binding bacteria by binding to Fe with an affinity higher than 10-12. BIOL 260 Lecture 5(3) Ed.1 - Apo- Tf is the iron-free form of transferrin that upon binding two Fe molecules, becomes diferric- Tf. This is accompanied by a conformational change to a cap-like molecule. This diferric transferrin binds to its receptor (TF- R) on the membrane and is endocytosed. - Due to the acidic nature of the endosome, the Fe molecules lose affinity to Transferrin, thus detaching from the latter and being retrieved into the cytosol where they bind to ferritin, an iron-binding protein inside the cell whose function is to deliver iron to proteins of the cell that need it for activation. Upon losing both Fe molecules, transferrin protein returns to original square confirmation. - Therefore, the TF-R is now occupied by the iron-free Apotransferrin. They go back to surface with receptor and are released to the outside. Unlike the previous pathways, here, the ligand and the receptor are retrieved (neither is degraded). Question: How does the cell sense the need for Iron? How is such a mechanism regulated? Answer: When iron is deficient inside the cell levels of Tf-receptor mRNA should be high (to bring in iron), whereas levels of Ferritin mRNA should be low (due to the low amount of iron inside the cell ). Now once the level of iron increases in the cytosol, the balance shifts and the cell will need more ferritin but less Tf receptors. This regulation by the cell is made possible through its possession of an iron-binding protein called Aconitase that can exist in two shapes: a. Rectangular when bound to Fe (Active Aconitase). b. Horseshoe in absence of Fe (Inactive Aconitase). In the absence of Fe, We must stabilize Tf-receptor mRNA and destabilize Ferretin mRNA. The horseshoe conformation is complementary to the hairpin loop structure found on different positions of both mRNA molecules. Binding of the horseshoe molecule to Tf-receptor mRNA prevents its degradation and thus i s stabilized. However the binding of same molecule to end of the Ferretin mRNA stops its translation and thus it is destabilized. In this way we have an increase of Tf- receptor expression and decrease in Ferretin expression. When iron builds up again, rectangular conformation is assumed and the Aconitase can no longer bind to mRNA. The reverse changes occur. Regulating mechanism at post-translational level. BIOL 260 Lecture 5(3) Ed.1 Fourth pathway: Transcytosis and IgG restricted to the breast and intestinal epithelia of ruminants (cows). - Figure 13-60 refers to an intestinal cell. IgG immunoglobulin is taken up by cell and is directed to early then late endosome. - Interestingly, the IgG escapes the lysosome and goes to the other end of the cell where it is released through the basement membrane into circulation resulting in systemic immunity. Another Example: Case of a Ruminant Breast - During lactation the IgGs follow the following pathway: basement membrane of breast epithelial cells transcytosed to the alveolar lumen intestinal lumen of suckling baby circulation. ruminants do not receive immunity through placenta unlike This results in the baby having the same immune profile as its mother. humans, but the immunity received by ruminants is systematic while that in humans is local. Note:Thefirst milk is called colostrum and is usually yellow due to high protein content. It is essential in ruminants since it is the source of systemic immunity for the baby. In case of humans, milk contains IgA  BIOL 260 Lecture 5(3) Ed.1 which only binds to cell surfaces and provides local immunity develops. Note: the exact sorting mechanism of IgG into milk that deviates it from lysosome is not clear. If well understood we could target transfected genes much more effectively. Extra notes: Endocytic cycles can play a role in cell locomotion (i.e. simply because exocytosis sites in certain cells as fibroblasts are not coincident therefore this can aid in cell locomotion). Cell Locomotion: For a cell to Endocytosis: This process move, it needs to extend its front involves the internalization edge (leading edge) and retract of membrane and its rear edge (trailing edge). The extracellular materials into leading edge often requires new the cell. It helps in recycling membrane material to form membrane components and protrusions like lamellipodia and receptors, which can then filopodia, which are essential for be reused at different movement. Exocytosis at the locations on the cell surface. leading edge supplies this new Exocytosis: This process membrane material. involves the fusion of Coordination: The non-coincident vesicles with the plasma sites of endocytosis and membrane to release their exocytosis mean that while the contents outside the cell. It leading edge is being supplied also adds new membrane with new membrane material material to specific areas of through exocytosis, the trailing the cell surface. edge can be recycling membrane components through endocytosis. This coordinated recycling and addition of membrane material help the cell to move efficiently Endocytosis is not necessarily the outcome of ligand receptor interaction.could trigger only a signal transduction pathway Important: If we were to inject foreign DNA into a cell, we must first coat it with calcium cations through a method known as calcium co precipitation technique. This will prevent the repulsion of DNA against the negatively charged cell membrane (due to NANA). After endocytosis, the DNA is inserted into an endosome and will reach the lysosome. Once there, a signal (cholesterol) must be added to the DNA molecule to tag it for retrieval into the cytoplasm and prevent its degradation (such as the case of cholesterol). Once in the cytosol, the DNA must be translocated into the nucleus, possibly by means of transport proteins and signaling peptides. Introduction to lecture six Intracellular sorting and trafficking: After mRNA is translated into proteins, the proteins have to be translocated to their respective destinations: