Biology 260 - Lecture 2 PDF

Biology 260 - Lecture 2 Summary of previous introductory session - T its microenvironment, including neighboring cells, the extracellular matrix (ECM) and the soluble mediators. - The cell has decided through time what is its optimal microenvironment for its function (in...

Biology 260 - Lecture 2 Summary of previous introductory session - T its microenvironment, including neighboring cells, the extracellular matrix (ECM) and the soluble mediators. - The cell has decided through time what is its optimal microenvironment for its function (in Cells have evolved over millions of years to thrive in specific microenvironments that best support their functions. This process of adaptation ensures that cells operate efficiently within their optimal conditions. In contrast, humans are still exploring and adapting to find the best environments for our well-being and productivity. This ongoing journey involves understanding our physical, emotional, and social needs to create environments where we can thrive. Where are the cells? Cells have localized functions, so they are not distributed randomly. Example One, the lung: - Epithelial cells lining the duct and bronchi are called the ductular epithelial cells. - Epithelial cells lining the alveoli are called alveolar epithelial cells. These cells serve in the exchange of gases. - Beneath these epithelia is the Extracellular Matrix (ECM); the ECM is made up of two parts: the basement membrane and the stroma. The basement membrane is the space between the epithelium and the stroma. The stroma is made of connective tissue. The ECM is made of collagen, proteoglycans and glycoproteins (produced by the cells), all of which interact with cells. - In the stroma, there are fibroblasts. Surrounding the stroma are the capillaries that are made of endothelial cells surrounding the lumen. In the lumen, there are red blood cells and white blood cells (immune cells). Immune cells can also be found dispersed in the ECM that surrounds the endothelial cells. The ECM is the space filling material. Beyond this ECM there are vascular muscle cells. - The smallest reproducible structure or basic unit found in every tissue and organ is: [Epithelial or endothelial cells basement membrane connective tissue (including fibroblasts)] - All the cells are organized in working communities that are held together by a mesh of protein networks made of proteoglycans, glycoproteins, etc. Side-Note: The basement membrane is NOT made of living cells. Example Two, the skin: - It consists of the epidermis and the dermis separated by the basement membrane. The epidermis is made of several types of epithelial cells, including keratinocytes. Keratinocytes are specialized epithelial cells whose primary function is the formation of a barrier against environmental damage through the expression of several structural proteins, such as  Biology 260 - Lecture 2 keratin the most abundant protein (Book: an intermediate filament protein). Keratinocytes pile on each other as the ones at the base (attached to the basement membrane) proliferate whereas the outer layers die and get sloughed off (Book: keratinocytes are not engulfed by neighboring cells). - The connective tissue (dermis) contains collagen. (Collagen skin products that people buy make no sense because they add collagen to the outside where it performs no function; you have to treat cells in order to produce collagen in the right place). Similarly, shampoo advertisements that claim to strengthen the hair through keratin treatment are unsound because strengthening the hair requires nurturing the cells from the inside, not outside (needs to produce its own microenvironment, its own collagen, etc.) - The sweat gland (sebaceous gland ducts) is also made up of epithelial cells that rest on a basement membrane. The epithelial cells produce sweat that is channeled out through a duct. Example Three, the breast: The breast consists of ductular epithelial cells, the alveolar structures and the ducts that lead to the tits, and the rest of the breast filling tissues is all ECM. The skin covering the breast tissue is another organ. When the woman is not lactating, the alveolar structures die out and are replaced by fat tissue. When lactating starts, the alveoli bud out and start producing milk. Example Four, bone: There are osteoblasts sitting on an ECM, which is highly calcified and consists of collagens that are cartilaginous in nature (Book: Osteoblasts are spread out but they are still interconnected. However, they are trapped in the matrix). There are different types of collagens in different tissues, which explains the case of calcified collagen in bones. Conclusion: All cells are attached to an ECM. Even sperm cells, eggs and circulating red and white blood cells were attached at an earlier stage of their differentiation in a tissue that follows the generic structure. Concept of dynamic and reciprocal interaction between the cell and its microenvironment: A cell needs to communicate with other cells in order to produce its ECM. This ECM in its turn tells the cell what to do by sending signals inward to the cell; there are receptors on the cell for ECM components. (It is also called dynamic reciprocity) - No organ is made of 100% cells. There are ducts, lumens and other cell-free spaces in each. Basement Membrane: This thin, non-cellular layer supports the epithelial cells. It is composed of proteins like collagen and laminin, providing structural support and anchoring the epithelial cells. Extracellular Matrix (ECM): Surrounding the ducts, the ECM is a network of proteins and other molecules that provide additional support and structure. It includes collagen, elastin, and proteoglycans. Connective Tissue: This tissue surrounds the ducts and contains various components like fibroblasts, blood vessels, and nerves. It provides further support and nourishment to the ductal cells Biology 260 - Lecture 2 How did all the specialized cells that make our body organs come to be? - Different location, different orientation, different position all deal with how cells became specialized for their roles. When the zygote attaches to its place in the fallopian tube and then in the uterus, one side of the cell, or later the blastula, is exposed to factors the other - The differentiation occurs not because the genome in each cell is changed, but because of epigenetic factors that lead to changing the shape of the DNA strands. For example, the promoter of a specific gene in cell X can be exposed to transcription factors due to histone acetylation whereas in cell Y the promoter might be methylated rendering it out of the cells and hence to different types. Acetylation: This process involves adding acetyl groups to histone proteins, which are proteins around which DNA is wrapped. Acetylation typically relaxes the chromatin structure, making the DNA more accessible to transcription factors and other machinery needed for gene expression. This usually results in enhanced gene expression. Extra information from the slides: Methylation: This involves adding methyl groups to DNA, usually at cytosine bases in CpG dinucleotides. Methylation often leads to a more condensed chromatin structure, which can block transcription factors There are different types of cells: from accessing the DNA. This generally results in repressed gene expression. Cells and tissue types in the stomach: Biology 260 Lecture 2 (continued) Edited The microenvironment was included to define the smallest functional unit in all the tissues within each and every organ. (. In all tissues (Breast, liver, intestine, skin, nervous system...etc) the smallest microenvironment consists basically of the: Three Functional cells (that produce the function) which are called parenchymal cells (but all cells are equally important). parenchymal cells are the cells of the organ that perform the main function of the organ like hepatocytes in the liver. Basal membrane sitting on a Stroma which has scattered fibroblasts in it. Note: blood capillaries which have the same microenvironment arrangement (endothelial cells, basal membrane, and their own stroma) pass through the stroma. Note: if we remove a cell from a lactating breast after it received the signals it will produce milk for a certain time then lose the function. (It needs basement membrane and other cells to produce milk) Note: the role of the basal membrane is not only for attachment, but the basal membrane and the neighboring cells also send signals as potent as hormone signals. Note: if we provide (in vitro) the right concentration of cells with the right substratum (not plastic but collagen for example) the cells will make their own basal membrane. Note: both the Basal membrane and the Stroma beneath it constitute the extracellular matrix (ECM). These are constituted of collagen, proteoglycans, and glycoproteins. (the ECM has fibroblasts on addition to these) 7 billion $ question: why do cells express different genes in each cell although they all have the same DNA? And what is the role of microenvironment in this? Gene Expression: Different types of cells (like muscle cells, nerve cells, etc.) express different sets of genes. This means that while the DNA is the same, only certain genes are “turned on” or active in each cell type. For example, a muscle cell will express genes that produce proteins necessary for muscle contraction, while a nerve cell will express genes that produce proteins necessary for transmitting nerve impulses1. Microenvironment: The microenvironment, which includes the surrounding cells, extracellular matrix, and signaling molecules, plays a crucial role in regulating gene expression. It provides specific signals that can activate or repress the expression of certain genes. For instance, cells in a particular tissue receive signals from their neighbors and the extracellular matrix that help determine which genes should be active Every cell has the same amount of DNA, which is 2 meters long packaged inside the nucleus which is 10 times smaller than the smallest thing you can see with the naked eye. Size of a typical cell is 10 microns -> 5 times smaller than the smallest thing you can see. Nucleus is 5 microns/ mitochondrion is 1 micron. 2 meters of DNA, 3 billion bases, containing 25000 genes packaged inside a 5 micron structure (nucleus) What determines if a gene is transcribed or not? 1. The architecture of the gene whether it is exposed or not (available for transcription or not) which is affected strongly by the methylation state of the DNA. 2. The presence of the right transcription factors Is the architecture of DNA in all cells similar? No, according to which genes are expressed in each tissue. But also in the same organ and the same tissue the DNA architecture is not similar in all cells (Why? Answer in the next question) Is the architecture of DNA in cells static or dynamic? Dynamic > architecture keeps changing with time. Even if they are very close to each other, their microenvironment might be extremely similar but there must be a difference. This difference might be due to a slight difference in the age of the cells. Note: cells are different on the per cell bases: the more distant the cells are > the more different And it is dynamic > continuously changing (making even close cells different from each other) Note: The DNA of a cell is undergoing constant remodeling so that every single cell at any time expresses about 3000-4000 genes only of its genome (25,000 genes).  How can the cell afford such a the cytoskeleton is composed of dynamic structure? 3 main protein structures that form a network which supports Cytoskeleton in the cytosole relays the cell structurally, relays signals within the cell mainly to the nucleus, allows intracellular signals to the DNA (but how?) and extracellular movement. these are: intermediate There are soluble signals, but it is not filaments, microtubules and microfilaments. the cell receives the main mediator There is a nuclear matrix inside the biomechanical and biochemical signals externally through surface receptors and relays them mechanically from the nucleus that is very similar to the cytoskeleton to the nucleus as the cytoskeleton is connected to the nuclear matrix through cytoskeleton > now called Nuclear nuclear membrane receptors called nuances. this relay of Skeleton. The Nuclear Skeleton is very signals regulates gene expression by exposing the similar to the cytoskeleton, and the DNA specific genes needed for expression according to the is attached to it. This structure is dynamic and it is the reason behind the dynamicity of the received signal. the nuclear skeleton is connected to the DNA and it is nuance dynamic which makes the DNA architecture dynamic too. (receptors that span the nuclear membrane and physically hook up the nuclear skeleton to the cytoskeleton) What is the cytoskeleton made up of? 1. Actin and Actin binding proteins 2. Microtubules and Microtubule associated proteins 3. Intermediate filaments and I.F. associated proteins. How can the cells afford the dynamic reorganization of the cytoskeleton? The Actin binding proteins (ABP), Microtubule associated proteins (MAP) and intermediate filaments associated proteins (IFAP) help the cell continuously arrange and structure the different filaments (the skeleton) in different ways (permutations) by putting them parallel to each other, anti-parallel, or crisscrossed. Note: Actin+ ABP+ Microtubules+ MAP+ intermediate filaments+ IFAP = dynamic cytoskeleton that is continuously changing and physically connected to the nuclear matrix that is physically connected to the DNA -> Continuum (e.g. If a cell moves to the right it will change the architecture of the cytoskeleton then its nuclear skeleton then its DNA structure then gene expression will change). In other words, there is an aspect of physical signaling between cells and not only chemical. Are neighboring cells connected? Yes, via junctions like Gap junctions, tight junctions and adhesion belts. And these junctions provide a continuum between one cell and the other because the junctions are hooked physically to the cytoskeleton (just like nuance on the nucleus). Also there is a connection between each cell and the basal membrane via receptors spanning the cell membrane called integrins that anchor the cell to the BM. Integrins mediate attachment but since the cytoskeleton is hooked to these structures it is no longer a physical attachment only but also a signaling modality as well. Note:. The constituents of the ECM (collagen, proteoglaycans, and glycoproteins) are as diverse as the constituents of cytoskeleton. These secreted proteins (by the cell) affect the structure of the ECM thus the cytoskeleton, the nuclear skeleton, the DNA architecture and evetually gene expression. Both the cytoskeleton and ECM are crucial for maintaining the structural integrity and functionality of cells and tissues. The cytoskeleton supports the cell internally, while the ECM provides external support and mediates interactions between cells. Note: (Actin+ microtubules+ Intermediate filaments in the cytoskeleton) = (Collagens + proteoglycans + glycoproteins in the ECM) The interaction between the cell and its microenvironment (ECM for example) is dynamic and reciprocal: the cell makes its ECM but later on this ECM tells the cell what to do via signaling. All these structures are responsible for exposing about 4000 genes at any one second to meet the cells demand -> they should be highly variable and differ between one cell and the other. If we take a specific cell in our body, it will have an organization of a nuclear skeleton number 170, cytoskeleton 7454, integrin type 10 and basement membrane organization 853. (these numbers resemble one of the permutations available resulting from different arrangements as we said above) And the cell next to it will have VERY similar organization since they are doing the same thing but will be slightly different (due to age difference as we mentioned). These permutations will keep changing with time because the cell keep on changing its surrounding and making new one. For example if we think of the parenchymal cells above as breast alveolar cells that are should not produce milk anymore -> cells shuts down milk production. Partly because of the physical pressure of the milk in the lumen, the paranchymal cells are pressed and they will stop making milk - These configurations will give us a specific DNA architecture. The BM is a dynamic structure. The cell changes it with time. ( ) There are 24 types of intergrins. integrins are a special type of receptors that connect the ECM to the cytoskeleton, allows Every cell from the diagrams receives signals from: bidirectional signaling between 1. Neighboring cells: cell-cell interaction. the cell and the ECM and plays 2. The ECM: cell-ECM signaling. crucial roles in cell migration, 3. Hormones, growth factors, cytokines, chemokines: Soluble signaling wound healing and immune responses. they bind to ECM These 3 parameters transfuse both physical and chemical signaling. proteins like collagen. Physical signaling: architectural changes Unfortunately, at the clinical level, when you go to the doctor and get your blood tested. The doctors look at the level of hormones. In other words the doctors only look at the third mode of signaling and only at the chemical part of it -> They look at only 1/6th of the parameters that affect your well being and diagnose you. Course Outline: 1. Different organelles inside a cell. 2. The cell as a whole 3. Cell-cell interaction. 4. Cell-matrix interaction. 5. Soluble signaling. 6. Organs 7. Cancer 8. Apoptosis  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure Introduction We all know that the cell membrane enclose the cell and marks its boundaries, but little we know about the other roles of the membrane. Ex. Signal transduction! The plasma membrane surrounds not only the cell as a whole but also its membrane- enclosed organelles. It is composed of dynamic1, fluid2 and thin (5 nm) lipid bilayers with protein molecules. The proteins in the bilayer have many functions due to their different structures; they act as transporters, ETC in mitochondria, receptors, recognition receptors (ex. MHCs), channels, NUANCE proteins, integrins, cell-cell junctions, enzymes etc. These MPs function mainly in signal transduction. The phospholipid bilayer contains different types of phospholipids giving it the aspects of fluidity and flexibility. Fluidity enables the phospholipid bilayer to act as a solvent for the membrane proteins, providing an optimal microenvironment for each protein to exist in, while flexibility will be defined by the ability of phospholipids to change their place at high rates3 to accommodate the dynamicity of proteins. We also know now that phospholipids are involved in signaling such as PIP2-IP3 signaling pathway, and triggering apoptosis. By this we introduce the idea that not only proteins are involved in signal transduction but also phospholipids. They relay signals in and outside of each organ. (Ex. mitochondria, NUANCE proteins in the nuclear matrix, ER, ribosomes etc.) Figure 10-1 1 Lipid bilayer is made up of varying amphipathic molecules. 2 Property of Phospholipids. 3 7 Some phospholipids change their place at a rate of 10 times /s  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure The Lipid Bilayer 4. This is a notion that proteins which are the other 50% play an incredible role in the plasma membrane. There are 3 types of lipid molecules in the membrane all of which are amphipathic molecules which simultaneously form bilayers: 1. Phospholipids. (somehow the most abundant) 2. Cholesterol. 3. Glycolipids. Phospholipids Mainly phospholipids are the most abundant component of the cell membrane. They have a polar head and 2 hydrocarbon chains of FAs that are 14-25 carbons in length. Usually one tail is saturated and the other is unsaturated with varying number of kinks5 (cis-double bonds). Fluidity of the membrane is controlled by the length and saturation rate of the chains because it affects the interaction between phospholipids. The longer and the more saturated the tail is, the more it can interact with the opposite tail, and the less fluid is the membrane. Amphipathic property of Phospholipids Figure 20-2 In an aqueous environment, phospholipids tend to simultaneously form bilayers or micelles. The heads tend to aggregate toward the aqueous environment while the tails towards each other. This simultaneous aggregation contributes to the healing properties of the bilayer. In case it was torn apart in a way, it will reseal itself. 4 Figure 10-7 By mass! The proportion of the number of proteins to phospholipids is 1:50. 5 1 or 2 are important and affect membrane fluidity.  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure A Two Dimensional Fluid It should be noted that in-vivo fluidity of the membrane is only controlled by the type of phospholipids as well their interactions with the proteins as will be explained ahead. Having said that, temperature has nothing to do with fluidity in-vivo given it is in the body. But still we tend to use in-vitro models with varying heat values to test the roles of phospholipids in membrane fluidity. For in-vitro studies, 2 models are used to learn more about the role of phospholipids in membrane fluidity, such as: 1. Lipid Bilayers: Liposomes: which is a spherical shaped vesicle used also in DNA transfection. Black membrane: a planar lipid bilayer which is placed across a hole between two compartments where it is used to test Figure 10-9b: Liposome. permeability of membranes. 2. Micelles complication: being studied without having cytoskeleton/cytosol. in vivo, lipid bilayer interacts with cytoskeleton. Figure 10-10: Black membrane These studying models and methods coupled with many others enabled us to learn more about the properties of phospholipids, such as their ability to rotate around their axis, movement, or even the effect of composition on the fluidity of the membrane. We were able to come up with the fluidity property of the membrane. We now know that there are 4 types of movement which are practiced by phospholipids: 1. Lateral Diffusion: (may reach 107 times/s)6 which is controlled by a reciprocal protein phospholipid interaction7. 2. Flexion: of the hydrocarbon tails, related to double bonds formation or breakage. 3. Rotation: perpendicular rotation across the axis. movement: regulates the microenvironment of the membrane proteins and hence affects their function 6 This movement dictates the movement of proteins which causes the movement of the cytoskeleton and thus affecting gene expression. 7 Affinity based interactions, will come up ahead.  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure 4. Flip Flop8: it as a movement which rarely occurs except with phospholipids in the SER where they are synthesized, and is used to move them from one monolayer to the other. Upon synthesis, phospholipid molecules are inserted in the cytosolic monolayer and then are moved from there to the other monolayer by an enzyme called phospholipid translocator (Flippase). To answer your questions on how the cellular membrane is made if there is no flippase except in the SER: Through studies we know now that as the phospholipid m diffuse to the ER, where they will move along with the budding vesicles from the Golgi9. Once the vesicle fuses with the plasma membrane, these newly Figure 10-11b synthesized molecules will move to join the plasma membrane. Then there is no need for any flip flopping in the synthesis of plasma membranes. In Addition, little is known about the synthesis of other membranes, such as the mitochondrial membrane. Note: it is important to mention also that the in-vitro studies at least till now are not able to study the protein-phospholipid interactions because we are never able to predict the exact composition of the phospholipids which are dictating the microenvironment of the proteins. For ex. We can test the effect of 10 phospholipid molecules on an insulin receptor in a synthetic model, but what about the remaining types of phospholipids, and suppose we knew their types how can we predict their exact position at specific time points? Super Dynamicity Model of Phospholipids With the various movement abilities of the phospholipid molecules, as well as its super dynamicity which can be recorded to span the whole bacterial cell in one second, two main questions are asked. What drives this movement? And why do we need them like this? Driven by Affinity, the phospholipids mainly move to accommodate the dynamicity of the proteins and to ensure the optimal microenvironment of the moving protein. Phospholipids move around to adjust the proper location of the protein at a specific time on the membrane ensuring its proper/optimal function. Also proteins are always moving, thus, for a protein to maintain its structure it requires a whole bunch of phospholipids around it. For ex. If proteins 8 More related to the synthesis and transport of the phospholipids in the SER. 9 Which are carrying proteins to be secreted, this vesicle are not mainly for phospholipids transfer.  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure changes their structure10 the phospholipids will have to change their structure too. This requires an incredible rate of movement. Note: the movement of the protein and the phospholipids is reciprocal and both induce each And the form of interaction is electrostatic, or chemical in case of a chemical bonding between the protein and the phospholipid molecule. Lipid Rafts There are certain regions on the membrane which we refer to as the Lipid Rafts and these are regions of phospholipids with long and saturated tails (less fluid). Found mainly in organelles and studied mostly in plasma membranes, these lipid rafts are the sites on the plasma membrane where most of the proteins are located. Figure 10-12 Notice in the figure the topology of the membrane from a top view. Red regions are lipid rafts. Figure 10-14a Figure 10-14b 10 As is the case when a receptor binds a hormone or when an integrin binds the ECM.  Biology 260 - Lecture 3 (1) Edition 2 Membrane Structure Proteins are found in rafts or in close proximity to these rafts, as a result we can deduce that the distribution of the proteins in the membrane is not random; they tend to aggregate in the vicinity of lipid rafts. Fluidity in the Membrane Depends on its Composition We measure fluidity of the membrane in the lab by the ability of the fluid to change from a t only occurs in in-vitro tests as mentioned before. The shorter the tail and the double bonds, the less there will be interactions between phospholipid molecules in a bilayer and the harder it will be for the membrane to freeze or gel. Biology 260 Lecture 3 (2) Edition 2 A two dimensional fluid: (Continued) Lipid rafts are the sites of accumulation of majority of the proteins in that vicinity. What is the importance of lipid rafts: There are a lot of lateral interactions between one receptor and another. Transactivation: A component of the ECM might bind to an integrin and activate a nearby receptor for insulin without having this receptor occupied by insulin. All these receptors are interlinked both physically and chemically. It will be discussed later in the course. Fluidity of Bilayer depends on its composition : Freezing point: It is an artificial temperature that we record fluidity by in the lab. It is the transition of the membrane from fluid state (highly motile) to the freezing state (the gelled membrane). This only happens in the lab, where we play with the temperature to observe such changes and it is called phase transition. This temperature depends on the composition of the membrane 1. The lesser the tendency of the phospholipids to interact with each other the harder it is to gel. This interaction depends on two factors: a) The length of the hydrocarbon chain: The shorter the tail the lesser is the tendency for interaction, the harder it is to gel (freeze) and hence the lower the freezing point. b) Cis double bonds (links): the more the double bonds the harder it is to freeze the membrane meaning it is more fluid, harder to freeze and hence has lower freezing point. 2. Cholesterol: Almost one molecule of cholesterol is found for each phospholipid molecule in eukaryotic cells. It is beneficial for us up to a certain limit. At the level of the head it is fixed.(proposed by a student ) It is slightly shorter than a typical phospholipid. The OH group is a polar head group that will fit in between two phospholipids. The amount of cholesterol in the membrane will afford the right fluidity for the cell. The cell always incorporates the right amount of cholesterol to ensure the right required fluidity. The correct amount of cholesterol is essential to avoid having brittle blood vessels arteries and capillaries. Biology 260 Lecture 3 (2) Edition 2 Cholesterol increases mechanical stability of the lipid bilayer and enhances its fluidity. Is it a controversy? Not really, because they are spatially distinct changes. From the figure, the ring structures are somehow rigid and when stuck between two phospholipids, this upper part of the membrane will become mechanically more stable. Whereas in the lower part of the membrane, the cholesterol tail inserts between the two phospholipid tails and renders them less available for interaction and therefore more fluid. (NB: question by student: how can things enter the cell if it is mechanically stable? Answer: nothing really enter the cell by diffusion unless small and hydrophobic, the cell is a highly impermeable structure and whatever goes in or out there are specific processes that we will be talking about during the semester. And this is true for all membranes, yet it is also flexible to accommodate for the physical signaling of the micro-environment) The cholesterol molecules also prevent the HC chains from coming together to crystallize thus making the membrane harder to freeze. Thus it enhances its flexibility such that it can still easily flip flop. Note: channels and other structures. Lipid bilayer as a solvent for membrane proteins: In the membrane there are different types of phospholipids and they are given their names depending on their head groups. The figure below shows the five major phospholipids ( at PH=7). Thus the lipid biological membrane consists of: 1. Cholesterol 2. Phosphatidylethanolamine 70% of bacterial membrane (no cholesterol is found. Cell wall replaces the need for cholesterol). 3. Phophatidylserine 4. Phophatidylcholine 5. Sphingomyelin (2-5 structural nomenclature of the head group of membrane phospholipid) 6. Inositol phospholipid (present in small quantities, very important in cell signaling PIP2-IP3 pathway). Each of these types have different subtypes, depending on the number (and position) of double bonds and the number of carbons in the fatty acid chains. Also the charge of the head domain differs between phospholipids, and it also depends on the PH of the microenvironment, and probably even nano-environment. (PH may vary between the nano environments of the membrane.) Biology 260 Lecture 3 (2) Edition 2 In the figure above, we see that Phosphatidyl Serine is negative at PH=7, whereas the rest are neutral at PH=7, if you lower the PH down to 6.8, the charge of the membrane may vary and this will affect the function of the protein. (The protein structure will also change because of the PH change.) Every single protein has the right combination of phospholipids around it, and every time a protein changes structure, the composition of the phospholipid in its vicinity changes. From the above table, we can see that the lipid composition of different cell types (and organelles) is different. The biological lipid membranes contain the above variety so as to serve as a solvent layer for the different membrane proteins. This composition will change depending on many factors, and thus it can be considered as a fingerprint for the composition of the phospholipid. These values are just approximations and averages taken at a specific time point of a specific population. (And if taken from vitro cultures they are meaningless.) Note: Membrane proteins are not necessarily functional in synthetic lipid bilayers as the latter contain specific types of phospholipids. I.e. for a membrane protein to be functional it requires the exact phospholipid micro-environment. Biology 260 Lecture 3 (2) Edition 2 Glycolipids are Found on Surface of Plasma Membrane - They are made of a lipid part made of different types of lipids (phospholipids, cholesterol) and a carbohydrate part, thus we know very little about them. - We know very little about these glycolipids - They are extremely variable. - They are about 5% of the total membrane lipids. - They are found on the non-cytosolic side of the membrane. They can be inside the lumen of the organelles so w. (It will become clear later why they are non-cytosolic.) - The function of such glycolipids found on the outer surface of the plasma membrane is to modulate how the cell perceives the ECM (ex: integrins), neighboring cells (ex: cadherin) or soluble ligands (ex: insulin {hormones}). Example: When insulin binds to its receptor, it will bind with certain affinity. If the receptor has a glycolipid next to it, the insulin will react with the receptor but it will be affected positively or negatively by the glycolipids. It may enhance or block the binding. The glycolipid that we know the most about is the NANA: N-acetylneuraminic acid (Sialic acid). NOTE BOX: Why do we know most about it? It is what gives a membrane a negative charge. So we studied it in order to help us with transfection (coating with CaCl2 Also, it helped us in culturing cells. We use tissue culture plates covered with Poly L-Lysine which is a positively charged amino acid so that when we add the cells to this plastic substratum the cells will stick better since the cells are negatively charged because of NANA. Biology 260 Lecture 3 (2) Edition 2 Lipid bilayer is Asymmetrical The asymmetry of the lipid bilayer is not a property of the proteins; it is a property of the phospholipids. The types of the phospholipid on one surface are different from the ones on the other surface. Both composition and charge are different and the significance of this difference is not yet clear. (Ex. Membrane bound protein kinase C is only active if the inner membrane surface is negatively charged). Interesting updates on Lipid Bilayer a) "Cell Membrane Reveals Surprising Organization": http://scicasts.com/bio/5324-cell- membrane-reveals-surprising-organization The publication: http://www.pnas.org/content/early/2013/01/24/1216585110.full.pdf b) "Pathway for Membrane Building Blocks" http://scicasts.com/bio/5338-pathway-for- membrane-building blocks?utm_source=feedburner&utm_medium=twitter&utm_campaign=Feed%3A+scicasts%2F news+%28Breaking+Sci-Tech+News+%7C+Scicasts%29 Lecture 4 (1) Edition 2 Membrane Proteins Membrane proteins associate with the lipid bilayer in five different ways Examples of membrane proteins: receptors for hormones, channels for transport, integrins that bind collagen, cadherins, transporters Generally, the membrane proteins can associate with the lipid bilayer in 3 different ways. (Most comprehensive) The eight are modifications of the 3 with 5 in-between major associations. The protein is either: I. Integral: spans the bilayer II. Peripheral: associates with bilayer via ionic interactions III. Covalently bond: via fatty acid linkage. The five major associations are: 1. -helix. Since the part traversing the membrane needs to be non-polar but peptide bonds are polar, therefore in the absence of H2O, the peptide bonds tend to form H bonds between them. Thus forming -helix structures (ß sheets is also possible) 2. -helices. They could also have a covalently attached fatty acid chain inserted in the cytoplasmic monolayer 3. Protein attached to bilayer by a covalently attached lipid. 4. Same as "3" but the attachment to the lipid is mediated via an oligosaccharide. 5. Some proteins attach to the membrane by non-covalent interactions with other membrane proteins (ex. peripheral membrane proteins). One protein pathway of the many pathways: ribosomes make protein from mRNA ER Golgi membrane. Lecture 4 (1) Edition 2 Take the protein on the right as an example: are: Disulfide bonds are on the non-cytosolic side of the protein and sulfhydryl groups on cytosolic side of the protein. When a protein is made and it s inserted in the ER membrane, it gets to a point where the transport stops, and the lower part of the protein will remain in the cytosol even though it will go later to Golgi and plasma membrane. It will stay there and thus disulfide bonds will not form. The other part of the protein is inside the ER and the disulfide bonds will form there and will remain formed even after it goes to Golgi and plasma membrane. Plus, glycosylation takes place in the ER and the Golgi, so the non-cytosolic part of the protein is the only part that may get glycosylated and never the cytosolic. There is typically a hydrophobic transmembrane alpha helical structure. (Other integral proteins can have beta sheets). It is always 21 amino acids because of the distance (one alpha helix). The amino acids are hydrophobic because of the hydrophobic environment of phospholipids and what are to the inside are the peptide bonds between the amino acids in which they are stacked away from the hydrophobic microenvironment of phospholipids. In this way it has to form the Alpha helical structure. Example: If you see a protein with 4 regions of 21 amino acids it means that it spans the membrane 4 times. (4 alpha helixes). C terminus is typically non cytosolic, the amino terminus is typically cytosolic. In oligomeric proteins covalent links are displayed on non-cytosolic sides and the oligosaccharides are on non-cytosolic part of protein. Again glycosylation occurs in ER and Golgi, and the part that will be inside the lumen of Golgi and ER (the non-cytosolic part) will be the site of addition of the oligosaccharides. Lecture 4 (2) Edition 2 III. Membrane Proteins: A. Membrane proteins associated with the lipid bilayer: The main pathway for deposition of membrane proteins on the membrane: mRNA is translated into a protein that is then inserted into the membrane of the ER, part of it being in the cytosol and another part in the lumen (or it is transported totally into the lumen in case it is not a transmembrane protein). Usually insertion is made in a way so that the N-terminus is located in the cytosol and the C-terminus is located in the lumen. The main sites of protein glycosylation are the lumens of the ER and Golgi apparatus where glycosylating enzymes are located. Once in the non-reducing environment of the ER lumen, oligomeric proteins will come together and disulfide bonds will form between two different protein subunits or within the same protein. A vesicle buds off from the ER such that the glycosylated side of the protein remains inside the vesicle. It moves towards and fuses with the Golgi apparatus (GA) membrane. In GA further glycosylation can occur. Then another vesicle buds off in the same manner as occurred in the ER and fuses with the cell membrane by exocytosis. NOTE: Other pathways will be discussed later (for mitochondrial or nuclear membranes, etc.) -helical structure of transmembrane proteins: The number of amino acids is always 21, all of which are hydrophobic. A multiple of 21 indicates the number of times the protein spans the membrane. Proteins that span the membrane several times are called If it spans the membrane an even number of times, then both the N and C termini are typically on the cytosolic side (there are a few examples where they are on the non-cytosolic side). -helices (or -sheets) since traversing the membrane needs to be non-polar but peptide bonds between the proteins are polar, so in the absence of H2O, the peptide bonds tend to form H bonds between them, hiding the hydrophilic part away from the hydrophobic microenvironment. Lecture 4 (2) Edition 2 Glycosylation: Cytosolic proteins and protein domains are never glycosylated (e.g. actin) while non-cytosolic proteins and protein domains are usually glycosylated (e.g. insulin receptor). Role of glycosyalation: 1. Targets proteins to their final destination by marking the glycosylated proteins as non-cytosolic. 2. Affects/ Modulates protein function. Different amounts of glycosylation confer a different function. B. Membrane Proteins can diffuse in the Plane of the Membrane: Significance of diffusion: o The protein needs to reach its final destination once it s produced. Examples include reaching the lipid rafts, dimerizing with other proteins. Also they need to move towards the proper glycosylating enzyme in the ER membrane (such as the one that adds fructose or mannose, etc ) since it is not the enzyme the moves towards the protein in this case. o Protein internalization by endocytosis occurs when the proteins are old or need to be removed (proteins need to clump together and form coated pits to be taken in by the cell). Modes of diffusion: o Rotational diffusion: Membrane proteins rotate around their axis (perpendicular to the plan of bilayer) which has to do with protein function. o Lateral diffusion which is an integral part of signal transduction. It is needed for dimerization, transactivation and more importantly for physical signaling by affecting cytoskeletal architecture. o Proteins do NOT flip flop. Lecture 4 (2) Edition 2 Questions concerning the speed of diffusion and whether a protein is stationary or not are significant in studying protein function. Techniques used to study lateral diffusion: 1. Fluorescence Recovery After Photo Bleaching (FRAP): It allows monitoring in real time the rate of diffusion of proteins on the membrane. - A membrane protein (ex: insulin) is GFP labeled and thus the entire cell surface becomes fluorescent. - A laser beam is used to bleach the signal of a certain area (i.e. proteins in this area are no longer florescent). - With time the florescence is recovered due to lateral diffusion of neighboring fluorescent receptors to the bleached area. - Faster recovery indicates a faster diffusion rate of the labeled protein. 2. Fluorescence Loss in Photobleaching (FLIP): FLIP is a modification of FRAP where a small area on the membrane is continuously bleached and fluorescence is measured in a neighbouring area. Fluorescence decreases in that area until the whole membrane is bleached, indicating movement of proteins in the membrane. The video played in class describes and illustrates the FRAP technique. In addition, a nuclear protein that is firmly anchored to the nuclear lamina and not able to diffuse was labeled, and the area showed no recovery after bleaching. Over time, the fluorescence in the neighboring area decreases as unbleached fluorescent molecules move into the bleached area and become bleached themselves. Complete Bleaching: This process continues until the entire membrane is bleached, indicating the movement and exchange of proteins across the membrane Lecture 4 (2) Edition 2 Green Fluorescence Protein (GFP): Expressed by jellyfish A scientist isolated the gene and fused it to an insulin-coding gene. (It can actually be fused to any suitable gene coding for a protein expressed on the membrane, and a strong promoter of choice can be added as well). Transfection of such a gene into mammalian cell allows the cell to produce a chimeric protein (protein-GFP hybrid) GFP does not usually affect the function of the protein it is fused to. Nevertheless, t functional. GFP labeling allows tracking the protein throughout the cell by video lapse photography (can be done in vivo). Drugs can be labeled and tracked as well. Later YFP (yellow) and CFP (Cyan) were isolated. Previously, radiolabeling was used and it required fixing the tissues and subjecting them to radiography. Thus, in vivo tracking was not possible. C. Membrane Proteins and Lipids can be Limited To A Specific Domain: The concept that the membrane is a lipid sea in which proteins float randomly is greatly oversimplified. It has some order in it. All membrane proteins end up on a specific location on the membrane (Basal, Apical, or Lateral). For example, an insulin receptor is always on the basal side of a cell and can diffuse within this region (with limited distribution) but cannot move to the apical side. This protein distribution is essential for its function and polarity. In the past they used to think that the cell-cell tight junctions prevent the movement of membrane proteins between apical and basal sides. But tight junctions themselves are proteins, so how are they localized as well? Distribution and localization of proteins is dictated by the phospholipid composition and its attachment to the cytoskeleton. In epithelial cells, tight junctions were once thought to solely prevent protein movement between apical and basal sides. However, tight junction proteins themselves are localized through interactions with specific lipids and the cytoskeleton, ensuring they form effective barriers.