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BIO241 Lecture 1-2

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University of Toronto St. George
Jennifer Harris

Tuesday, January 6, 2008 - We have 1 midterm, 1 final exam and 6 labs. Labs are 27% with 8% from quizzes, best 2 out of 3 (4% each). 65% is from exams: the midterm is 25% and covers section 1 of lectures and labs 1 to 2. The final exam is worth 40% and covers all lectures and labs 1-6. - Midterm is different from course manual: it is actually on Friday February 27 from 2 to 4. - There will be lab tutorials that will be held in 3 weeks, there will be 3 tutorials, one before the midterm and two before then, they appear on alternating weeks on Mondays from 3 to 4 and will be posted next lecture. - If you have any questions, asked the professor after lecture, come to tutorials or ask on the course website on Blackboard. He doesn’t take questions during the lecture. - This is a picture of a cell and the central dogma of biology is that DNA encodes mRNA which gets translated into proteins. You go from DNA to proteins and proteins are functional components of living cells. - We’re used to seeing pictures like this where the cell is static and doesn’t move but even in a simple diagram like this, you can see there will be lot of movement – first to transcribe DNA into mRNA there needs to be proteins on the DNA moving along the DNA & transcribing the DNA into mRNA. Then the mRNA needs to be transported out of the nucleus and into the cytosol. In the cytosol is where ribosomes bind to mRNA and starts the translation into proteins. That is movement again, movement of mRNA outside of nucleus and into the cytosol where ribosomes will move onto the mRNA, move along it and translate it into proteins and the proteins will move around the cell, some will be secreted outside the cell going through the ER. - This is all to emphasize that there is a lot of movement within a cell & from this picture, one can appreciate that there are numerous compartments within the cell so the cell is compartmentalized into different functional sub compartments like the nucleus that stores DNA & is also the main site for transcription. Then you have the cytosol & different organelles in the cytosol such as ER that all have specialized functions. The cell is compartmentalized in order to provide functional specialization & there is a lot of movement in the cell that is not captured in images such as the one in the slide. - There is a lot of movement in the cell whether it's proteins moving, whether it's signals being transmitted from the outside of the cell to the inside of the cell or whether it's the movement of proteins or lipids within the membranes that surround the cell or the organelles within the cell. - A movie is playing emphasizing movement within the cell. - We will appreciate how active it is inside the cell eventually and we’ll appreciate more the little subtleties we saw in the movie as we go through this part of the course. - Here is an animal cell which has a number of compartments in it called organelles and we’re familiar with these compartments such as lysosome which is the site for the degradation of what the cell doesn’t need anymore, proteins or structural components, that it doesn’t need will go to the lysosome and be degraded. - The mitochondria are the energy source for the cell where ATP is produced. - The nucleus is where the genetic material DNA is stored and site of transcription from DNA to RNA. - The Golgi is the site of protein modifications and sorting. - The endoplasmic reticulum – you have two types: you have the rough ER that is transcribing transmembrane proteins and proteins that will be secreted outside the cell surrounding these little specks called ribosomes that are actively translating proteins from mRNA & delivering them into the ER. You also have smooth ER which is a major site of lipids in the cell. - You can see all these compartments that are separated from each other have specialized roles in the cell. - He has highlighted 2 compartments or properties of the cell: the extracellular matrix and the lysosomes that are specific to animal cells – the extracellular matrix plays an important role in the adhesion of the cell to a specific location & also plays an important role in maintaining cell shape & another role in cell development. - In contrast, plant cells have some specific organelles that aren’t mainly found in animal cells – a major one are chloroplasts that are the site of photosynthesis and are found only in plants and not animal cells. - A vacuole plays a prominent role in a plant cell – they are mainly composed of this large vacuole and there are 2 types of vacuoles: one acts like a lysosome that is found in animal cells and the other ones are for storage of proteins and certain small molecules and they can actually be storage spaces for antimicrobial compounds so when the cell gets punctured, the compounds ooze out of the cell and will kill whatever is trying to invade it. - Okay so you have a vacuole, chloroplast and importantly a cell wall surrounds plant cells and not animal cells – the cell wall is a rigid structure and makes the cell often look square so it is involved in maintaining cell shape as well as help protect from external stresses such as temperature, mechanical stress, insect invasion or bacterial invasion so the cell wall surrounds the plant cell. - Importantly, of course plant cells also have structures such as a nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes (the site of degradation of many fatty acids), mitochondria and plasma membranes and are also found in animal cells – the ones listed in the slide are in both animal and plant cells. - There are lots of different compartments with functional specialization. - Cytoplasm is the space outside the nucleus, everything outside the nucleus in the cell shown in the slide. The cytoplasm would include the organelles also. - The cytosol is the aqueous component of the cytoplasm so it doesn’t include the organelles. - The lumen is the aqueous part inside organelles – so inside the mitochondria there for example, inside the organelles is the lumen. - In section 3 we will cover the membrane components, so the membranes that define the different organelles within the cell & actually make the boundary of the cell by the plasma membrane. So what are the components of these membranes that delimit the organelles & the cell itself? – that is the 1 st lecture. - Then there is a conundrum b/c if you separate your cell from the environment, you need ways of transporting small molecules or getting signals across from one side of the membrane to the other so we are going to look at how cells can transport small molecules across these membranes in both organelles & the plasma membrane (Lectures 2-4). - Lectures 5-6 we’re going to look at protein sorting so how do proteins get to different places in the cell – they all start out on ribosomes in the cytoplasm & then get shuttled to different compartments of the cell. Some proteins will end up in the membrane, some will be secreted outside the cell & others may be retained within the cell. Just to emphasize, it all starts in the nucleus DNA  mRNA  cytoplasm where it is translated into proteins and that is where it initiates. - Vesicle trafficking will be lectures 7-8 so how do things get around the cell in vesicles – so basically one important thing is things that are destined for membranes or outside the cell must be translocated through vesicles – they will start out in the ER, so these are transmembrane proteins or things destined for the outside of the cell, & will be translocated into the ER, through the Golgi apparatus & then shuttled to their appropriate compartment in vesicles. - Exocytosis & endocytosis is the final part of vesicle trafficking. One of the destinations of vesicle trafficking is exocytosis, translocation of things outside the cell & we’ll also cover how things get into the cells through endocytosis, so how do cells take up molecules from the outside? - Finally we’ll go into a different type of movement which is the transduction of signals from the outside of the cell to the inside of the cell – this should read lectures 10, 11, 12 & not 13 in the slide, so lectures 10 – 12 will be signal transduction or transmission of signals from outside of the cell to the inside of the cell. - You see there is movement that will occur at all these different parts of the section 1 lectures. - We start with membrane structure – they delimit the cells & organelles. - Cell membranes enclose the cell and the organelles – it is an important thing to keep in mind because this is what allows the different compartments to maintain their functional distinction from the rest of the cell so you have division of the cell into compartments and this defines the boundaries of these compartments and maintains differences b/w the cytosol, the organelles & the extracellular environment. These differences are what’s required to maintain different functions in each of these different compartments. - It is important to realize that it is the cell membranes that allow the cell and the organelles to maintain these differences. - To emphasize again, this is not always obvious, so where are these cell membranes localized? It is important to realize that it isn’t just the plasma membrane that is a cell membrane. - The first top-left arrow points at the plasma membrane and then there is the nucleus that is also surrounded by a membrane (going counterclockwise in the diagram). - The ER is also surrounded by a membrane. - Here is the Golgi, Golgi stacks would be surrounded by membrane, different compartments again. - Then there is a lysosome for example that would be surrounded by a membrane. - The vesicles here that would be budding off the Golgi are also surrounded by membranes. - The mitochondria and the chloroplasts in plants would all be surrounded by membrane so you can see that cell membranes are found throughout the cell and it's not only limited to the plasma membrane. - So again the cell membranes divide the cell into compartments & control the movement of molecules b/w these different compartments. - There are 2 major components to all these cell membranes: one is a lipid bilayer, and the lipid bilayer is the basic unit of each of these cell membranes. This is shown in the cross-section and what that means is that there are 2 leaflets to each bilayer, an outer leaflet and an inner leaflet – each one of them is a bilayer of lipids and this structure is very fluid, meaning it's not a static structure, things can move around within this layer. The PM of a mammalian cell has the viscosity roughly equal to olive oil so you can imagine that it is quite fluid & things could move around within this lipid bilayer. - This is the structural component of cell membranes & the functional components are mainly carried out by membrane proteins & they’re shown in the slide in green in the cross-section of the lipid bilayer. These proteins can be transport proteins that allow the movement of small molecules across the membranes b/c again, a lot of these cell membranes will divide the compartments & keep specific molecules out of these compartments & then these membranes can have proteins that selectively allow specific molecules to enter into these compartments – this is carried out by transport proteins. - Then there is the transmission of signals from outside the cell to the inside of the cell and is carried out by transmembrane receptors, these would be proteins found in the lipid bilayer that would allow for transmission of a signal from the outside of the cell to the inside of the cell. - Membrane proteins can also be receptors that recognize specific molecules outside the cell & allow the cell to respond inside. - The structural component again is the lipid bilayer and the lipids that compose the bilayer are called amphiphilic which means they have both hydrophilic or a polar head group (which is the ball structure) and also have a hydrophobic or non-polar tail. - What happens is that these amphiphilic molecules will associate spontaneously into a lipid bilayer facing their hydrophobic tails towards one another and the hydrophilic or polar head groups towards the aqueous environment which is found either outside or inside the cell (the cytosol) – basically the environment of a cell is aqueous. What happens to these amphiphilic lipid molecules is they point their fatty acid tails away from the aqueous environment and point their head groups towards the aqueous environment forming the lipid bilayer. - That basic structure of lipid bilayers in the cell. - We will look at for example, since it's a lipid bilayer, there are 2 faces to it and if we look at the plasma membrane, there is one side of the plasma membrane that will be facing the cytosol which is called the cytosolic face and the other side of the lipid bilayer is called exoplasmic face so these are the lipids with their head groups facing the outside of the cell. You have the cytosolic face facing the cytosol – these would be the head groups facing the cytosol and the exoplasmic face facing the outside of the cell. - Every lipid bilayer has this property, even if we look at the Golgi, you’ll have a lipid bilayer and in red there, it would be the cytosolic face because this lipid bilayer is facing the cytosol and the brown layer which is facing the lumen of the Golgi would be the luminal face of this lipid bilayer. - These lipid bilayers in cells are mainly composed of phospholipids so they have a polar head group and two hydrophobic tails shown. - There are many different categories of phospholipids & in an aqueous environment, these phospholipids will spontaneously form/self associate into a bilayer. If you drop these phospholipids into water for example, they would spontaneously form a lipid bilayer with tails facing each other & the hydrophilic heads facing the aqueous environment or water. This would be called an artificial bilayer. In water if you drop these phospholipids, they will form a bilayer. - We mentioned that lipid membranes are fluid – that means that lipids can move. This movement is limited, lipids can move in lateral diffusion so they can move from side to side – this is very rapid so lipids can move very rapidly within one leaflet, they can also flex so they can move their tails back and forth & they can also rotate but notice that all of these movements are within one leaflet – lateral diffusion is within one leaflet, flexion is within one leaflet and rotation is within one leaflet. - Flip-flop from one leaflet to another is very rare and it doesn’t happen spontaneously very often, the movement of one lipid from one leaflet to another is a very rare event and this is what is called a flip flop. - The rate of movement within a lipid bilayer or within one of the leaflets of the lipid bilayer depends on the structure of the lipid. - Animation: it shows different structures will move at different rates. Description: you have 2 different lipids, you can see the fatty acid chains, there are fatty acids that are straight saturated fatty acid and unsaturated fatty acid chains have little kinks in it. You can see from the movie that different types of lipids would move at different rates within this lipid layer – the saturated ones move slower but the unsaturated move faster. The structure of these lipids determines how rapidly they move within the leaflet of the lipid bilayer. - Although it doesn’t happen spontaneously, there will be flip-flop of lipids from one leaflet to another. There are enzymes in cell membranes that carry out this function of flipping one lipid from one leaflet to the other leaflet. - An example of this is phospholipids that are synthesized on the cytosolic leaflet of the ER need a phospholipid translocator that will rapidly flip-flop the lipid that is newly synthesized on the cytosolic side to the noncytosolic side. - You can see there would be a problem if there wasn’t this flip flop where this lipid bilayer would be growing only on one leaflet and then you wouldn’t be able to grow the lipid bilayer so what happens is that since all of the lipids are synthesized on the cytosolic side of the ER, once it’s synthesized on the cytosolic side, there is an enzyme catalyzing the flip flop of one of these lipids to the noncytosolic leaflet. This evens things out so that there will be equal distribution of lipids on the cytosolic side and on the luminal side of the ER. This is why these flip flops are needed and one of the reasons is that phospholipids are only synthesized on one side of the ER. - Just to go into the lipid components in more detail, we mentioned that a major component of cell membranes is phospholipids – these are the most abundant in cell membranes and an example of these is phosphatidylcholine shown in the slide. - We aren’t going to go into the structure of these phospholipids but you can see there is the polar hydrophilic head group and two non-polar hydrophobic tails or fatty acid tails that may be saturated or partially unsaturated fatty acid tails so this is phosphatidylcholine, there is also sphinomyelin, phosphatidylserine, these are all examples of phospholipids. - The phospholipid composition is important for the function of cell membranes because some membrane proteins require specific phospholipids for their function, either they will be activated by specific phospholipids or they will bind to specific phospholipids so they need specific phospholipids in that membrane in order to function so we’ll see the different membranes in the cell will have different composition of phospholipids and that can be very important for protein function. - Another lipid component of cell membranes are steroids. In animals, the major steroid would be cholesterol shown in the slide. It has a rigid steroid ring structure & a little polar head group & a non-polar hydrocarbon tail which allows cholesterol to insert itself into a lipid leaflet as shown in the slide so the hydrophobic polar head group can interact with the polar head groups of the phospholipids & then the steroid ring & the non-polar hydrocarbon tail can insert itself into the hydrophobic component of the lipid leaflet. - In animals, cholesterol is the major type of steroid but in plants, cholesterol is very rare – instead there are many plant specific steroids found in membranes of plants. - In animal cells, cholesterol and phospholipids can be up to 1:1 ratio and we all know cholesterol b/c if it builds up too much in your system, it can cause heart problems. - One of the major roles or the major properties of cholesterol in lipid membranes is that it decreases the mobility of phospholipids. We saw that phospholipids can move in a leaflet but if there is a lot of cholesterol in that leaflet, then it decreases the mobility of these phospholipids & makes the plasma membrane less permeable to molecules that can normally can traverse the plasma membrane. - Overall, cholesterol makes lipid bilayers more rigid & decreases mobility of phospholipidsnd it also decreases the permeability of the plasma membrane. That is the 2 major lipid component of cell membranes. - Glycolipids are the 3 major component of cell membranes and as implied are sugar groups attached to lipid molecules. - You can see the lipid molecule and this particular one has a galactose sugar attached onto it. - Lipids are glycosylated in the lumen of the Golgi apparatus, that is where the sugar will be added onto lipids & then glycolipids are found in the plasma membrane & very rarely but on some intracellular membranes. They are synthesized in the Golgi & most of them end up in the plasma membrane & about 5% of the total lipids in the outer leaflet of the plasma membrane are actually glycolipids in animal cells. - We say the outer leaflet and this will become more apparent also since the sugars are added on the luminal side of the Golgi apparatus, the sugars or these glycolipids will end up on the extracellular face of the plasma membrane. Basically, anything that will be on the luminal side of the Golgi will end up on the outer leaflet of the plasma membrane. - Glycolipids are on the outer leaflet of the plasma membrane, their synthesis is on the luminal face of the Golgi & they are involved with the interaction of the cell with the environment & they protect also from harsh conditions. So they have very general roles and they are important roles but they’re mainly found on the extracellular face of the plasma membrane. - This brings up an important point where the leaflet, the bilayer of a cell membrane is nonuniform, there is asymmetrical distribution of different lipids within the membrane. We just mentioned that glycolipids are on the exoplasmic or the outer leaflet of the plasma membrane but you also have phospholipids such as phosphatidylserine shown on the slide that has negative charges & that is found on the cytosolic face of the plasma membrane. This is important because it plays an important role in signaling. - One protein called protein kinase C actually binds to phosphatidylserine and is activated by the phosphatidylserine and therefore requires this specific phospholipid to be present on the cytoplasmic side of the plasma membrane in order to be activated. - The plasma membrane & the lipid bilayers in general are not uniform in terms of which phospholipids or just lipids in general are found on the outer or inner leaflet – there is an asymmetrical distribution of lipids within the lipid bilayer. - That is the structural component of cell membranes, the 2 component & the functional component of cell membranes are membrane proteins. - The membrane proteins provide the specific functions of certain membranes surrounding organelles or the cell & they can associate with the lipid bilayers in a number of different fashions highlighted in the slide. - The first way is transmembrane proteins. - Transmembrane proteins transverse the entire lipid bilayer as shown in the slide and are also called amphiphilic so that means again, they have a hydrophilic domain which is aqueous so that hydrophilic domain will be exposed to the outside or inside of the cell, the cytosol, that is the aqueous environment & they also have a hydrophobic component & this is the membrane spanning domain that will make contacts with the hydrophobic chains of the lipids that make up the bilayer. Amphiphilic means they have both hydrophilic & hydrophobic components to them. - You have different types of transmembrane proteins – you can have a single pass transmembrane proteins and this is basically made up of one alpha helix that will traverse the lipid bilayer. You have multi-pass that have multiple alpha helices that traverse the lipid bilayer & you can also have some beta barrels that will be more rigid & also traverse the lipid bilayer. - Most of these are glycosylated – if these are glycosylated then they’re glycosylated also on the luminal side of the Golgi, mainly in the Golgi and if they’re glycosylated on the luminal side of the Golgi, the glycosylated residues of the transmembrane proteins will be exoplasmic or facing the outside of the cell – this is the same as for the glycolipids added on the luminal side of the Golgi, they also face the outside of the cell. - Many transmembrane proteins are glycosylated & the sugar residues are added in the lumen of the Golgi & therefore the sugars will face the outside of the cell. - A different way of associating with membranes is anchoring with one amphiphilic alpha helix on one side of the lipid bilayer so one leaflet of the lipid bilayer. So this alpha helix here is actually amphiphilic itself so one side of the alpha helix is hydrophobic associating with the inside of the lipid bilayer & the other side is hydrophilic & interacting with the aqueous environment of the cytosol. Here within one alpha helix, this alpha helix is amphiphilic and has both a hydrophobic and hydrophilic component & this protein can then associate with the lipid bilayer with one leaflet. - This is in contrast to transmembrane proteins that would completely traverse the lipid bilayer. - You can also get lipid anchored membrane proteins. This instead of the protein traversing the plasma membrane, they are actually modified in the cytosol by the addition of a fatty acid chain or a prenyl anchor. These are both hydrophobic anchors that can then anchor the protein in the lipid bilayer, as shown in the slide. This is the protein in the slide and this would be either a fatty acid or prenyl anchor. - Now for different types, a myristoyl is a type of fatty acid anchor that is added to the N terminal glycine of proteins. A palmitate is a fatty acid anchor that is added to internal cysteine & a prenyl anchor (different types are farnesyl or geranylgeranyl anchors) that are added to a C terminal cysteine. - You can see the different anchors are added to different locations on a protein. Both types are synthesized in the cytosol, this is important b/c transmembrane proteins are synthesized in the ER – those will associate with the PM or the cell membrane by traversing it completely whereas these lipid anchored membrane proteins are synthesized in the cytosol & their anchors are added in the cytosol & then they will associate with their appropriate membranes. - Both types will be directed to the cytosolic face. The protein will be synthesized in the cytosol, the anchor is added & this protein dissociates with the cytosolic face of the lipid bilayer. - These are shown just to give you an idea of what the structures look like – you don’t need to memorize what the specific structures of these are but mainly to know where they are associated in the protein. - The myristyl anchor or the myristate anchor is added to the N terminal glycine and will anchor the protein (peace sign protein). - Then there is the palmitoyl anchor that will anchor from an internal cysteine & you can see that this would be the sulfur group from that cysteine that would be associated with the palmitoyl anchor & that will anchor it into the membrane. - Then a farnesyl anchor that has a slightly different structure but the important thing is again you have that sulfur group from the cysteine but this time it is a C terminal cysteine on the protein now that the farnesyl anchor will be associated with. - N terminal glycine, internal cysteine and C terminal cysteine is where the anchors are added in these specific cases & then all of them will end up on the cytosolic face of the lipid bilayer. - Another example is the GPI anchor. This is another anchor that can be added to proteins & this will allow it to associate with the one leaflet of the lipid bilayer shown in the slide. This would be the GPI anchor that is associated with the protein. - Importantly, this one is added to proteins in the ER. You’ll get the GPI synthesized in the ER & then it will be added onto the protein. Then it will end up on the cell surface so again, synthesized inside the compartment of the ER & ends up on the surface of the cell. - This was the same for glycolipids & glycoproteins – if you get a modified on the luminal side of an organelle, it ends up on the surface of the cell. - Finally, you have proteins that are not directly associating with the membranes but they’re bound to either face to proteins that are associated with the membrane, with the modifications or through the mechanisms just covered. These are all called peripheral membrane proteins, so you would have a transmembrane protein and then you would have a peripheral membrane protein that is associated with the membrane protein on either face, it could be either the exoplasmic or the cytoplasmic face of the cell where these peripheral membrane proteins could associate with other membrane proteins. - One of the differences is that these are mainly non-covalent interactions that are occurring with these other proteins that are integral membrane proteins & for a biochemist that means that these proteins are much easier to extract from a cell & that is one of the key features of the different types of proteins and is that these peripheral proteins are easier to extract from cells than for example, fatty acid modified proteins or especially transmembrane proteins. - Peripheral membrane proteins only need a gentle extraction procedure that doesn’t destroy the lipid bilayer to extract it from the cell. - This is in contrast to integral membrane proteins which in order to be extracted from the cell, you need to be able to destroy the membrane with strong
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