Cell Diff Test 1

Nucleic acids, proteins, and polysaccharides are __________________--

marcomolecules - macromolecules are polymers or chains of subunits connected by covalent bonds - the length of a given macromolecule may vary considerably, from just a few subunits, to many of thousands, in the case of some polysaccharides, and millions in the case of DNA that makes up a single chromosome

Lesson 4

membrane structure - Cell membranes such as the plasma membrane serve to delineate the boundary of the cell, and in the case of eukaryotic cells, serve to create internal compartments that can have different chemistries and functions within the overall structure of the cell - The plasma membrane is also the primary interphase through which cells interact with the extracellular environment

The bilayer creates a sealed compartment with no edges .

- A lipid bilayer separates an inner environment from an outer environment, this is because the bilayer creates a sealed compartment with no free edges, as shown in the Left Fig. - A planer phospholipid bilayer would require that phospholipids on the edges be exposed to water, which is an energetically unfavorable situation, instead a continuous sheet is formed which created an internal aqueous environment separated from an external aqueous environment - Right Fig. depicts a cross sectional view of a continuous bilayer. - Many images of the bilayer show the phospholipids on the edge of the sheath, but keep in mind this is just for simplicity and would never naturally occur

Compartmentalization of eukaryotic cells

- A membrane separate the lumen of each compartment from the surrounding cytosol -Organelle membranes separate the inside compartment (lumen) from the cellular material outside of each organelle (cytosol) - Membranes that form the internal compartments of eukaryotes act to create chemical and functional difference between the inside of each compartment and the outside of the compartment, much like the plasma membrane acts to create differences between the inside of the cell and the outside of the cell - Cytosol: cellular material inside the cell membrane, but outside the membrane bound compartments and is highlighted in blue in Fig B. "soluble" material enclosed by PM but outside of compartments - Material that makes us the cytosol is often referred to as soluble - Cytoplasm: All the cellular material enclosed by the plasma membrane (PM) - includes cytosol and compartments (everything inside PM)

Ion gradients can power active transport

- ATP or light powered - Most active transporters or pumps can be classified as uniporters, meaning they transport one molecule in one direction as shown at the left in the Fig, these are typically powered by energy provided ATP or light - Couple transporters in contrast are powered by energy stored in an ion gradient, there are two types of couple transporters defined by the relative direction of movement of the molecules being transported - For simp orders shown at center, both molecules are moving in the same direction, the co transported ion in this case is the one providing the energy by moving down its concentration gradient, and this movement powers transport of the red molecule u its concentration gradient - N contrast, for the antiporter shown on the right, the molecule being transported moves in the opposite direction of the co transported molecule - Antiporters are often referred to as exchangers because effectively one molecule is being exchanged for another

Transport protein connections to disease

1: Cystic Fibrosis - Most common mutation is deletion of Phe508 in chloride channel protein - Protein is misfolded and degraded - Defective chloride transport => ion inbalance creates thick mucus and makes it more difficult to clear bacteria from lungs 2: Multidrug Resistance (MDR) transporters - Pump hydrophobic molecules out of cells - - While it is hopefully obvious membrane transport proteins are critical for cellular function, there are several different situations in which specific defects in transport proteins have been connected to a human disease. One example involves the development of cystic fibrosis, one common cause of this disease is associated with a deletion of a singe amino acid Phe508, in a chloride channel protein. This mutation causes the protein to become misfolded and is then subsequently degraded. The absence of the protein alters chloride ion balance in lung tissue which creates extremely thick mucus in the lungs making it more difficult for the patient to clear bacteria from the lungs. Individuals who suffer from cystic fibrosis often die because of resulting bacterial infections. Another example involves the multidrug resistance or MDR transporter, these proteins are normally involved in pumping hydrophobic molecules out of cells, usually that is fine, but if cancer cells acquire the ability to overexpress MDR proteins then they become resistant to chemotherapy drugs, many of which are hydrophobic, in fact chemotherapy treatment can actually select for cancer cells that are able to overexpress MDR protein, because such cells are protected from the drugs and will survive

Lesson 5

Membrane transport

Other transporters carry out active transport to create and/or maintain gradients

- Active transporters require energy input to transport or "pump" molecules up/against their concentration gradient -While some transporters work by passive transport, others work by active transport - Cells typically use active transport to create and maintain a concentration gradient - Unlike passive transport, during active transport, the molecule being transported is moving from the low concentration side to the high concentration side, which requires the input of energy since such movement is energetically unfavorable. That is the solute or cargo is essentially being transported or pumped up or against its concentration gradient -A given transporter is either a passive transporter or an active transporter, it cannot be both, - The energy input for active transport comes from a variety of sources, the most common energy source is the hydrolysis of ATP to ADP and inorganic phosphate as shown in the center example. Certain transporters instead use energy from light, in which light is absorbed and used by these proteins to pump their cargo, but these are much less common - Another example is the couple transporter shown on the left, which utilizes energy released by one molecule moving down its concentration gradient to a different molecule up its concentration gradient - In all three examples the molecule indicated by the orange square is being transported up or against its gradient

Primary Cells

- Advantages • Because they have been recently removed from the organism they may be more life-like and may exhibit more life-like properties may exhibit some activities of differentiated cell, thus if one wants to approximate a cells productivity within an organism, primary cells are as close as one can get - Disadvantages • are mortal, have a limited experimental timeframe • Since they are derived from tissues that usually contain a mix of cell types, one must often conduct experiments on a mix population of cells, so interpretation of experimental results may not always be straight forward • difficult to store, they generally don't survive freezing or other techniques designed to preserve the cells . This can cause problems for cell reproduction as well

Membrane transport proteins are either transporters and channel proteins

- All are multipass proteins => create hydrophilic path through lipid bilayer - There are two major classes of membrane transport proteins, transporters and channel proteins. All membrane transport proteins are multipath proteins, this allows the protein to create a hydrophilic path through the hydrophobic core of the lipid bilayer. - Transporters and channel proteins function by different mechanisms. Transporters function to move a solute that is a cargo molecule by what is referred to as a ping pong mechanism. In this process the cargo binding site opens on one side of the membrane, the cargo molecule is bound by the protein and then the protein subsequently undergoes a conformational change that exposes the binding site to the other side and the cargo is released to this side of the membrane - Transporters by definition bind to cargo molecules, they have specific binding sites to which these molecules must bind and often binding is what triggers the conformational change of the protein. Because they have a binding site they are typically specific for a given cargo, in that only a particular cargo can fit in the binding site - In comparison channel proteins function by creating a pore in the membrane, channel proteins therefore do not bind to cargo. However, depending on the amino acids that line the pore and the diameter of the pore, channel proteins can still be relatively specific - In the Fig it shows an ion moving from one side of the membrane to the other through the pore of the channel protein

The best understood channel proteins are ion channels

- All channel proteins work by passive transport => molecules move down concentration gradient - For channel proteins, the best understood are the ion channels, which as the name implies it will allow for the movement of ions from one side of the membrane to the other - Some channel proteins are always open, these are referred to as leak channels because the ions present on one side of the membrane can leak through the channel protein to the other side - Other types of channel protein open and close in response to some sort of signal, these are referred to as gated channels, and as the name implies, they poses a gate that is either open or closed. If the gate is closed then the ion can't move through the channel - All channel proteins work by passive transport, meaning the ions move down their concentration gradient from the side where they are abundant in concentration to the side where they are relatively rare in concentration, so there is no energy input associated with movement through channel proteins which is the defining property of passive transport

Transcellular transport

- An important physiological example where several transport proteins work together to internalize glucose for the gut into the body occurs during transcellular transport. In this example intestinal epithelial cells which line the gut control glucose uptake by the asymmetric distribution of different transport proteins. On the apical surface which faces the intestinal lumen, shown in orange in the diagram, there are sodium driven glucose symporters. These proteins move glucose up its concentration gradient from the intestinal lumen into the cytosol of the intestinal epithelial cells. The energy input for the movement of glucose is provided by the movement of sodium ions down their concentration gradient from outside the cell to inside the cell. Thus glucose import into the intestinal epithelial cells occurs via active transport, once glucose is in the cytosol the intestinal epithelial cells will then need to move glucose outside of the cell into the extracellular fluid where it can diffuse and travel throughout the body to be used by other cells. In transcellular transport glucose it relatively high in concentration in the cytosol compared to the extracellular fluid. On the basal lateral surface, which is the portion of the intestinal epithelial cell that faces the extra cellular fluid, there are paths of glucose transported. On this surface, glucose just moves through released proteins by the process of facilitated diffusion to enter the extracellular fluid. Notice these cells also have the sodium-potassium-ATPase, labeled here as the sodium pump, which is critical for maintaining the sodium gradient across the plasma membrane and thus provides the energy source to run the sodium driven glucose symporters

Cell lines

- Are immortal: have an unlimited reproductive capacity • Does NOT mean they will never die! - Cell lines are Generated or Obtained from: •Biopsies of tumors. The classic example is the helia- cell line, which was isolated from a biopsy of cervical carcinoma taken with permission from Henrietta Lax in the late 1950's. Helia cells are one of the most commonly studied cells and is currently grown in laboratories around the world • Stem cells, like cancer cells these are immortal. Cell lines derived from adult stem cells are relatively common, but those from embryonic stem cells are tightly regulated • treat primary cells with mutagens: involves temporary exposure of primary cells to carcinogens, this is to induce mutations that would convert a mortal cell into an immortal cell • spontaneous mutations in primary cells may occur in cultures that would convert a mortal cell into an immortal cell (in rodents, not humans) - In an attempt to overcome some of the limitations when working with primary cells, many researchers instead work with cell lines. - Cell lines are cultured cells that descended from a single cell and thus all the resulting cells are in a direct line from that original cell - The main advantage of cell lines is that they are immortal, which in this context means they exhibit an unlimited reproductive capacity. When cell lines are grown in culture cell number constantly increases and never plateaus as what is seen for primary cells. Immortal does not mean the cells will never die, individual cells in a culture dish die all the time, but there are always new cells ready to replace the dead ones - Advantages in addition to being immortal: • Because they are a direct descendant of a single cell, they are homogeneous (contain only one type of cell), and thus all cells in the population are identical. A researcher dealing with and epithelial cell line, knows that they are working only with that cell type • survive freezing, so they can be stored long-term and shipped to other labs around the world • All these advantages provide better confidence in the reproducibility of the results over time and between different research groups - Disadvantages • have mutations: other than stem cells, all sources of cell line involve abnormal cells and any experiment must be interpreted in light of such abnormality. The helia cells mentioned previously commonly contain almost twice the chromosomes as a normal human cell. Even for stem cells the age of the culture may be an issue, as many stem cell lines accumulate defects as they proliferate in culture. • may accumulate more mutations in culture • must interpret any experimental results in terms of mutations .

Peripheral membrane proteins bind to TM protein or phospholipid head

- Can be removed with agents that disrupt protein-protein or protein-lipid noncovalent interactions (low pH or high salt solutions) - Peripheral membrane proteins can interact with either a transmembrane protein as shown in this fig, or with the head of a phospholipid. In either case, these interactions are noncovalent and therefore peripheral proteins can be removed from the membrane with agents that disrupt protein-protein or in some cases, protein-lipid noncovalent interactions. Typically such treatments involve solutions of either low pH or high salt so that if a cell is treated with a high salt solution for example any peripheral membrane proteins on the extracellular face of the plasma membrane would be displaced and could be subsequently isolated

Phospholipid provide the fundamental structure of cellular membranes

- Cell membranes are composed of different types of molecules, but the most foundational are phospholipids, which provide the fundamental structure for all cell membranes. - Phospholipid molecules form a structure known as the lipid bilayer which is depicted as a simple horizontal gray bar in this fig. Despite the simple representation, it would be impossible to have a cell membrane without phospholipids and all other molecules that associate with the cell membrane do so by interacting with phospholipids, either directly or indirectly

All cells must routinely transport hydrophilic / large molecules across membranes

- Cells use transport proteins - Lipid bilayers serve as selective barriers to movement of different molecules based on the chemical properties of those molecules - Small hydrophobic molecules can readily diffuse across the bilayer and small uncharged polar molecules can diffuse to some extent. The larger polar molecules or ions are unable to diffuse across the membrane to any degree or even at all - All cells must routinely transport hydrophilic and large molecules across membranes - In the fig on the right, in the stylized cell ions have to moved both in and out of cells across the plasma membrane, nutrients such as glucose as well as the building blocks for macromolecule synthesis also have to be brought in from across the membrane, molecules also need to move into and out of the membrane enclosed organelles of the eukaryotic cells. For example ATP is a charged molecule and thus the ATP produced in mitochondria must somehow be moved out of the mitochondria and into the cytosol so it can be used as an energy source four the reactions necessary for life. At the same time pyruvate and ATP must be imported into the mitochondria so that this organelle can continue to make more ATP - so cells have to be able to move molecules that can not simply diffuse across a bilayer for all kinds of purposes. In order to do this they need help, this comes in the form of transport proteins (one of the main functional classes of membrane proteins)

Ca++ transport proteins in the ER membrane

- Cytosol: 10-7 M Ca2+ - ER/SR lumen: 10-4 to 10-3 M Ca2 - Calcium transport is also critical for the endoplasmic reticulum, in this case the primary transporter is calcium atpase which is essentially the same protein as described in the plasma membrane in the previous slide, however we are now looking at the endoplasmic reticulum membrane rater than the plasma membrane, so keep in mind the at cytosol is above the ER membrane indicated by the horizontal gray lines and the lumen of the ER is below. In this case calcium is being transported out of the cytosol into the lumen of the ER and the concentration difference is substantial, 0.1 millimolar in the cytosol, as indicated in the previous slide, and 0.1-1 millimolar in the lumen of the ER. Similar to the plasma membrane, calcium atpase, for each atp hydrolyzed one calcium ion is transported out of the cytosol, in this case, into the lumen of the ER

common features of the 3 domains of life; Bacteria, archaea, and eukaryota

- DNA is the genetic material - Express genetic information via similar mechanisms - a plasma membrane separates the interior of the cell from the extracellular environment - examples of the same metabolic processes are found in all the three domains

Detergents are able to break apart cell membranes

- Detergents are amphipathic. - Nonionic detergents are gentle and allow proteins to remain functional - ionic detergents are harsh and will denature proteins - Integral membrane proteins can only be isolated if the membrane is destroyed. How does one go about destroying the membrane in order to isolate transmembrane proteins or other types of integral proteins. The answer is that one uses detergents. It turns out that detergents are amphipathic molecules that can have a hydrophilic head group and a hydrophobic tail group, much like phospholipids, and can readily insert into a lipid bilayer - In this diagram detergent molecules are the little orange lollipop like structures - Assume we are interested in isolating the transmembrane protein, shown in green, from the plasma membrane of the cell. Upon addition of detergent, the detergent molecules are able to insert into the lipid bilayer and fragment the membrane into little pieces. Some of the detergent molecules will associate with the transmembrane domain, that is the hydrophobic portion of the protein that normally passes through the bilayer. The really interesting thing is that if one next removes the detergent and adds back phospholipids, an artificial lipid bilayer will reform, a protein will insert into that bilayer and the function of the protein can be further studied. If one wants to reconstitute functional proteins, one would use a nonionic detergent, which is considered gentle because it will not disrupt protein function. - In comparison, ionic detergents are relatively harsh and will denature the protein so that any function will be lost

All phospholipids are amphipathic and have same fundamental structure .

- Different types of phospholipids are present in cell membranes by all share the same fundamental structure. A polar/hydrophilic head attached to nonpolar/hydrophobic tails - This Fig. shows four different representations of the phospholipid phosphatidylcholine. The most common type of representation for all phospholipids is the simple lollipop with two sticks, shown at left. The fatty-acid tails are hydrocarbon chains that can vary in length and may contain double bonds that create kinks in the chain structure - Molecules like phospholipids that contain both hydrophilic and hydrophobic regions are termed amphipathic. As we will see, this chemical property is critical for phospholipid function and for membrane formation

The amino acid sequence provides information for correct folding

- Each different type of protein has a different amino acid sequence, it turns out that the amino acid sequence provides the information to determine the correct folding protein into the correct tertiary structure - In this Fig. we look at some classic experiments performed in the 1960's. The enzyme rnase was placed in a test tube with appropriate buffer conditions. The enzyme was in its native tertiary structure (native in this case means the enzyme was active, it was folded correctly and couldn't carry out the degradation of RNA). When the purified RNAse was subjected to heat and treatment with a chemical called a reducing agent, the protein unfolded, the heat disrupted the noncovalent bonds that hold the protein in its tertiary structure and the reducing agent acted to break disulfide bonds, which are indicated by the orange lines between the cystines, which are indicated in turn by the numbered blue dots. Following this treatment the protein unfolded into its denatured state, that is the primary structure of the protein, and the protein as expected, had no activity. If the solution containing the protein was cooled down and the reducing agent was removed after a period of time, the protein would refold into its native tertiary structure and regain the ability to degrade RNA, thus the protein regained its structure and consequently regained its activity. In this case the only information that was available to allow the denatured protein to fold back into its native structure was the amino acid sequence of the protein, hence the conclusion that all information for the correct folding of a protein is found in that proteins amino acid sequence

Membrane asymmetry: carbohydrates and proteins

- Each face has • different proteins • different domains of TM proteins - The glycocalyx is a prime example of membrane asymmetry, since the carbohydrates associated with the glycocalyx act to protect the cell and are involved in cell-cell recognition, it makes sense that these membrane components would be found on the extracellular face, but not the intracellular face of the plasma membrane. However, membrane asymmetry is not limited to carbohydrates or types of phospholipids - Proteins also exhibit an asymmetrical distribution at the plasma membrane and in fact every internal membrane also has an asymmetric distribution of proteins - In this diagram of the plasma membrane, note that different proteins are exposed on one face or the other and that for transmembrane proteins different domains of those transmembrane proteins are exposed on one face or the other. Such asymmetry goes back to the fact that the function of the two sides of any membrane are different and therefor it makes sense that the composition of the two sides of any membrane would also be different - If you look at the absorbed glycoprotein on the extracellular face of this membrane, there is no counterpart on the inside, since that protein is presumably supposed to function on the extracellular face. Perhaps in cell recognition or some other process that the cell uses to interact with the extracellular environment

Cell membranes are asymmetric .

- Each face has a different function and a different phospholipid composition - Most cell membranes are comprised of 4 main types of phospholipids. In addition, the number of phospholipids on one face, is essentially equivalent to that of the other face. However, the type of phospholipid is not equivalent on each face, this makes cell membranes asymmetric in terms of the phospholipid distribution. Thus, one face is different than the other in terms of chemistry, and therefore function. - In the case of the plasma membrane example shown here, the extracellular face is composed primarily of PC and SM, while the interface is composed predominantly of PE and negatively charged PS

All proteins are polymers of amino acids

- Each type of amino acid has a different side chain (R group) - The structure of an amino acid is shown in the fig. it consists of a central carbon with 4 chemical groups, the amino and carboxylic acid groups that give the amino acid its name, a hydrogen atom, and an R group or side chain that distinguished each type of amino acid. - The amino acid group and the carboxylic acid group carry a positive and negative charge respectively at neutral pH, which is the situation for most proteins

Ca++ transport proteins in the plasma membrane

- Environment 10-3 M Ca2+ - Cytosol 10-7 M Ca2+ - Active transporters powered by ATP hydrolysis of ion gradients allow for the movement of a wide variety of molecules across both the plasma membrane and internal membranes - The movement of calcium is particularly important in cell signaling and cells generally try to keep a relative low concentration of calcium in the cytosol by using a number of different transporters - The typical cytosolic concentration of calcium is about 10^7 M or 0.1 mM. in comparison the environmental concentration of calcium for a typical animal cell is around 1 millimolar which is roughly a 10,000 fold difference in calcium ion concentration, in order to maintain this difference cells use a number of transport proteins in the plasma membrane to pump calcium out of the cell, one of these is the calcium ATPase. For each molecule of ATP hydrolyzed one calcium ion is pumped from the cytosol into the extracellular environment - Cells also use a calcium sodium exchanger, here sodium comes down its concentration gradient from outside the cell and the energy released from the movement of sodium provides the energy to move one calcium ion from the inside to the outside

small changes in protein structure can produce major changes in protein activity

- Every protein, whether it folds correctly by itself or with the assistance of chaperon proteins, it exists for some period of time before it is denatured one last time and is degraded back down to its component amino acids. During its lifetime a protein may continuously undergo small changes in its structure while retaining its general overall shape. Although such structural changes may be small, they can often have major impacts on the activity of the protein. In fact this is a major way in which protein activity is regulated (that is turned on or turned off). This particular example, we are looking at the structure of the G protein. G proteins are able to bind GTP and hydrolyze GTP. The right fig shows two different structures for the protein depending on whether the protein is bound to GTP (top panel) or hydrolyzes that GTP and therefor had bound GDP (bottom panel). In this case the G protein is involved in translation and whether GTP is present or whether GDP is present determines whether a transfer RNA molecule can bind to the protein. Notice the structural change that occurs on the right hand diagram depending on whether GTP is bound or GDP is bound. The structural changes influence the activity of the protein, such structural changes, even though they are relatively small, have a major impact on the activity of the protein. These structural changes in turn influence the activity of the protein and even when such changes are small and temporary, they are able to have a big impact on the proteins ability to do work.

Many proteins require assistance of chaperone proteins to fold correctly

- Example: Hsp70 prevents misfolding & facilitates some folding - Although the experiments in the previous slide described that a proteins amino acid sequence provides the information to determine a proteins tertiary structure, subsequent experiments revealed that many proteins require further assistance to fold correctly. This assistance comes in the form of chaperone proteins. Chaperone proteins do not determine the final structure of a protein, instead they facilitate the folding process and allow the protein to establish its correct 3D structure. A number of chaperone proteins fall into the category of proteins called heat shock proteins (HSP). Heat shock proteins were discovered when cells were heated to a slightly elevated temperature and scientists observed that certain proteins increase in abundance in response to this heat shock. Various types of Heat shock proteins have since been identified, these include HSP70's and HSP60's in bacteria. Note that there are eukaryotic equivalences of these proteins as well. HSP70's function is to prevent misfolding and facilitate some folding of the newly synthesized protein. In this Fig. we see HSP70 (shown in red) binding to a newly synthesized protein, this newly synthesized protein is kept form folding incorrectly by the presence of the HSP70's. in some secondary and perhaps tertiary structure is also generated while the chaperone proteins are bound to the new protein.

Quaternary Structure

- Formed by interaction between two or more polypeptides (subunits) - Generated by same types of bonds that produce tertiary structure - Although some polypeptides are able to carry out functional roles once tertiary structure is achieved, other must interact with other folded polypeptides in order to become functional. Such proteins or multi subunit complexes form what is known as quaternary structure, the highest level of protein structure, just like different domains may have functional roles within a folded polypeptide, different subunits in turn have different functional roles within a multi subunit complex. The number and identity of subunits varies from complex to complex. Some proteins are composed of only 2 subunits, while multi subunit complexes like the ribosome may be composed of dozens. The interactions that hold two or more polypeptides together to form quaternary structure include the same types that generate tertiary structure, hydrogen bonds, ionic bonds, disulfide bonds and hydrophobic interactions.

The Glycocalyx

- Glycoproteins are mostly protein - Proteoglycans are mostly sugar - Glycolipids are modified lipids - The carbohydrates on the outer face of the plasma membrane form a structure called the glycocalyx, which essentially means sugar coat. This sugarcoat or carbohydrate layer can e visualized by electron microscopy - In the lower figure, the gray haze denoted by the two arrows is the glycocalyx - In the fig on top we see a cartoon image of the glycocalyx, the carbohydrates that form the glycocalyx are attached to the proteins and phospholipids that are exposed on the outer face of the plasma membrane. The glycocalyx is composed of number of different types of molecules - Glycoproteins are proteins that have been modified with various carbohydrate groups, but by mass are mostly proteins - Protoglycans tend to have more elongated, extensive sugar chains and by mass are mostly sugar. - Glycoplipds are phospholipids or other lipid molecules in which the hit group has been replaced with or modified by a sugar chain of some sort

H+ transport proteins in the plasma membrane and lysosomes membrane

- H+ transport controls pH of cytosol and lysosome lumen - Cytosol pH 7.0-7.2 - Transport ions are also critical for regulating pH, both in the cytosol and lumen of lysosomes. In the case of the cytosol, one important protein on the plasma membrane is the sodium proton exchanger. Here sodium moving down its concentration gradient from outside the cell provides the energy to pump a proton from the cytosol into the extracellular environment. The cytosol in most cells is relatively neutral with a pH of around 7-7.2, which is critical for the functioning of cytosolic proteins and other molecules. In the case of the lysosome, the interior of the lysosome is much more acidic than the cytosol in order to facilitate the ability of these organelles to carry out the degradation of macromolecules, here cells use a proton ATPase to actively pump protons into the lysosomal lumen in order to reduce the pH to around 5. This transporter pumps 1 proton into the lysosome for each molecule of ATP hydrolyzed

Hydrophobic interactions contribute to protein folding

- In this Fig. we see an unfolded polypeptide with nonpolar sidechains indicated in green, and polar sidechains indicated in blue, ,yellow, and red. Typically as shown at right, when a protein folds, the hydrophobic, nonpolar side chains tend to cluster in the middle of the protein and are effectively buried away from the interaction with water molecules. In comparison, the polar side chains tend to exposed on the outside of the folded protein where they form hydrogen bonds with the surrounding water molecules.

Every type of protein has a unique amino sequence

- In this slide we see the amino acid sequence for the protein alpha-tubulin, specified by the single letter amino acid abbreviations. Amino acid number onathyamine is at the N terminus and amino acid number 451 nutiroscene is at the C terminus - A proteins amino acid sequence is its ultimate identifying feature and the only way to be absolutely sure that one protein molecule is different from another protein molecule is to determine the amino acid sequence of each

Functional classes of membrane proteins

- Membrane proteins can be classified by function, and the major functional classes are: Transporters and channels which help small molecules move across lipid bilayers, anchor proteins which can attach a membrane to either the extracellular matrix outside the cell or to the cytoskeleton inside the cell, receptor proteins which allow cells to recognize signal molecules, and various enzymes that are important for membrane specific functions. In all cases the proteins describes are drawn as penetrating through the membrane, this will ring us to another way to classify membrane proteins, that is how proteins associate with the membrane

_________________ allowed scientists to visualize cells and structures within cells and became one of the approaches used by scientists to classify organism

- Microscopy - we recognize 3 domains of life: Bacteria, Archaea, and Eukaryotes - at the cellular level we can refer to bacterial cells, archaeal cells, and eukaryotic cells, which all exhibit several common features.

Membrane proteins are either integral or peripheral.

- Must disrupt membrane structure to isolate integral membrane proteins - Can strip peripheral membrane proteins from membrane without damaging membrane structure - Membrane proteins are either integral or peripheral. - In this Fig. Various proteins are shown in green or gray, keep in mind whenever we are looking at drawings of the plasma membrane, by convention, the extracellular environment side is on the top, and the cytosolic side is on the bottom. - Of the example proteins shown in the figure, A,B, and C represent integral proteins and D represents a peripheral membrane protein. - As the name implies, integral membrane proteins are an integral part of the lipid bilayer in fact in order to isolate integral membrane proteins, one must actually destroy the membrane structure - Transmembrane proteins in example A pass directly through the lipid bilayer and typically have a region of the protein exposed on each side of the membrane - Some transmembrane proteins pass through the membrane only one time, while other transmembrane proteins pass through the membrane multiple times - Other integral proteins as shown in example B are embedded in only one of the two phases of the bilayer and are therefore exposed to only one side of the membrane - Example C depicts the third type of integral protein, which is connected to the membrane through covalent attachment to a membrane lipid or fatty acid anchor and thus does not pass into or through the lipid bilayer - Peripheral proteins as shown in example D, whether on the outside or inside of the cell are associated with a transmembrane protein indicated in gray. By definition peripheral proteins can be removed from the membrane without damaging the membrane structure

Functions of membrane bound compartments

- Nucleus: protects DNA from chemical and physical harm, and physically separates the process of RNA synthesis (transcription occurring in the nucleus) from protein synthesis (translation that occurs in the cytosol). This separation means the processes of transcription and translation may be independently regulated, a level of control not possible in prokaryotes. This is the defining internal compartment that distinguishes eukaryotes from prokaryotes - Mitochondria: the primary site of ATP synthesis. Often referred to as the ATP factory of the cell - The ER: the major site of protein and lipid synthesis, and delivers these products to the golgi apparatus. It is a network of tubes and sacs spread throughout the cell interior - Golgi apparatus: Modifies the ER products and sorts these products to their working location. These are critical components of the interconnected path that includes vesicles, the plasma membrane, and the extracellular environment. Is a relatively defined stack of compartments usually located in the nucleus - Lysosomes: Degrade complex molecules into building blocks that can be used by the cell to make new molecules. Macromolecules are broken down into building blocks that the cell can reuse in future biosynthetic reactions - peroxisomes: provide environment for detoxification reactions involving hydrogen peroxide that acts to neutralize toxic chemicals - plant cells would also contain chloroplast as well as a multipurpose organelle called the vacuole,which are located within the cell wall.

X-ray crystallography is main approach used to determine protein structure

- Often most difficult part is forming cystal. - Must know amino acid sequence - Now facilitated by use of robotics - Scientists are not yet able to predict a polypeptide bonds 3D structure simply by looking at its amino acid sequence, instead the tertiary structure of the folded protein must be determined empirically, although effort intensive, this is critical information given the inherent relationship between structure and function, - the most common approach used to determine protein structure is X-ray crystallography. In this approach a scientist must first generate a crystal of the purified protein, next a beam of x-rays is directed on to the protein crystal and the resulting diffraction of x-rays is recorded as a so called diffraction pattern, which is unique to the specific protein structure. The diffraction pattern along with the amino acid sequence of the protein is fed into a computer program which deduces the proteins 3D structure. Historically one of the most difficult parts of this procedure was finding the correct conditions to get the protein of interest to form crystals, but now much of this procedure has been sped up by the use of robotics, even so, we still know much more about the primary structure of proteins than either tertiary or quaternary protein structures

Measuring membrane fluidity by fluorescence recovery after photobleaching

- One approach to quantifying membrane fluidity is termed FRAP of fluorescence recovery after photobleaching. In this approach fluorescent molecules are used to label cell surface proteins or phospholipids. A region of the fluorescently labeled is then eradiated with a laser which bleaches or destroys the fluorescence thus creating a bleached region. Overtime fluorescent molecules recover, in that they move back into the bleach spot while some of the bleach molecules move away from this region. We can monitor recovery of fluorescence into the bleached region, and an example is shown in the graph. Following bleaching there is a small decrease in fluorescence in the irradiated region. Overtime fluorescence recovers back into that region and a rate of recovery is an indication of the relative mobility of the molecule that was photobleached. Typically, phospholipids, because they are smaller, will recover much faster than larger proteins, likewise proteins that are complex to other proteins will take longer to recover than single protein molecules. Whether recovery occurs completely to the 100% level, or not, depends on what the labeled molecules interact with. For example, if label proteins are stuck to either the cytoskeleton or the extracellular matrix that may prevent complete recovery

The The Na+K+ ATPase is an essential plasma membrane transporter

- One of the most important transporters in animal cells is the sodium-potassium-ATPase, this transporter uses energy from ATP hydrolysis to move sodium out of the cell and potassium into the cell. In doing so the Na+K+ ATPase creates and maintains both a sodium gradient and a potassium gradient across the plasma membrane - For each molecule of ATP that is hydrolyzed, free sodium ions are pumped out so that there is much more sodium outside of the cell than inside the cell. At the same time two potassium ions are pumping up their concentration gradient from outside the cell to inside the cell, so there is much more potassium inside the animal cell than outside. The relative importance of the sodium - potassium - ATPase for animal cells is indicted by the fact that depending on cell type this protein utilizes roughly 1/3 - 2/3 or all the ATP produced by the cell, so cells are putting quite a but of energy into creating and maintaining the sodium and potassium gradience across the plasma membrane. Tuns out this is not the only consequence of this proteins activity.

The Na+K+ ATPase helps regulate animal cell volume

- Osmosis is the passive movement of water from high to low concentration - Occurs partially by diffusion through bilayer but mostly through aquaporins (channel proteins) - inibition of Na+K+ ATPase will burst plasma membrane - Turns out that The Na+K+ ATPase also helps to regulate animal cell volume - Movement of water molecules from high concentration to low concentration is termed osmosis - For most cells there is a high concentration of water outside the cell than inside, while the opposite is true in terms of solute concentration. Water molecules tend to move down their concentration gradient into the cell. Some of this movement is simple diffusion through the lipid bilayer of the plasma membrane. The rate of water movement via this mechanism is slow due to the fact that water is polar. To allow for more significant water movement into the cells, which are aqueous environments, cells posses channel proteins called aquaporins to facilitate water movement across the plasma membrane. The overall movement of water is a delicate balance and cells use a variety of mechanisms to maintain their volume, if too little water flows inward they will shrivel, too much and they will burst - Here on the left we see an animal cell with a typical lower concentration of solutes outside compared to the inside of the cell. We know that the Na+K+ ATPase is involved in volume regulation because treatment of cells with drugs that inhibit this protein lead to increased inward flow of water by osmosis cause the cell to swell and eventually burst as shown in the figure - the Na+K+ ATPase contribution to maintaining cell volume has to do with the fact that one net solute is pumped out for each ATP hydrolyzed, so the protein is constantly working to reduce the internal solute concentration and the osmotic force to pull water into the cell - Living organism have evolved different ways to deal with the inward movement of water - Certain protozoa expel water through an organelle termed the contractile vacuole and plants keep their cells from bursting by having a strong cell wall

Some chaperones create chamber to facilitate correct protein folding

- Other chaperone proteins act to create a chamber to facilitate the folding process for newly synthesized proteins, such chaperons include the HSP60's (mentioned previously) - There the newly synthesized protein is inserted into a chamber and under these protected conditions is able to find its correct folded structure. As with the previous example the chaperone protein in this Fig. is simply facilitating the folding process, it is not directing the new protein how to fold

Membranes are two-dimensional fluids

- Phospholipids and cholesterol move within plane of membrane due to relatively weak hydrophobic interactions - lateral diffusion: 2- Dimensional movement - Spontaneous movement from one face to the other is Energetically unfavorable - In addition to being asymmetric, cell membranes also represent two dimensional fluids in which phospholipids and cholesterol molecules can diffuse laterally within one of the two faces of the bilayer. Such movement is possible due to the noncovalent interactions between molecules, which can rapidly break and reform - Individual molecules can likewise flex and rotate within one face or the other, however phospholipids and cholesterol molecules almost never spontaneously move from one face to the other, a behavior known as flipflop. Such movement is energetically unfavorable because the hydrophilic head would have to pass through the hydrophobic core of the bilayer - Membrane fluidity is critical for membrane function and cells may adjust the chemistry of the phospholipids and the amount of cholesterol in order to maintain fluidity, especially in the face of lower temperatures - Generally speaking, shorter hydrocarbon tails and unsaturated tails with a greater number of double bonds will increase number and fluidity

Membranes are two-dimensional fluids

- Phospholipids, cholesterol and proteins can move within membrane due to relatively weak hydrophobic interactions - Membrane proteins are also subject to the 2D fluid properties of cell membranes - In this classic experiment, a human cell and a mouse cell were fused together to create a hybrid cell. Prior to the fusion event, membrane proteins on the extracellular surface of the mouse cell were labeled with a red fluorescent dye, where as membrane proteins on the extracellular surface of the human cell were labeled with a green fluorescent dye. Following cell fusion the hybrid cell had initially had a region of green and a region of red, but overtime these regions mixed together, so that at about 40 mins after cell fusion there was a relatively homogenous distribution of both red and green molecules, indicating that some of the green molecules diffused across to the initial red side and vice versa. - Similar techniques have been used to demonstrate that proteins as well as phospholipids and cholesterol all move within the membrane and that such movement is due to relatively week hydrophobic interactions that are strong enough to keep the molecules within the membrane, but week enough to allow molecules to change positions or diffuse within the plane of the membrane

Protein misfolding and disease

- Prion diseases: Creutzfeldt-Jakob Disease, Kuru, Scrapie, Mad Cow Disease -A protein must be folded correctly in order to function, at minimum if a protein is not folded correctly then it will not function correctly and the cell would lose the function of that protein. Other problems can result from failure of a protein to fold correctly. For example instead of just being denatured or unfolded the protein could misfold and thereby create other problems for the cell, in fact protein misfolding is tied to a number of different diseases - In the Fig. we are looking at a protein called PrPC. If there is a mutation in the gene for this protein that can create amino acid changes in the protein sequence, certain of these mutations lead to a different version of the protein, which is called PrPCSe. This mutant protein not only folds incorrectly, but actually folds in such a way that hydrophobic regions of the protein are exposed on its outer surface, in an aqueous environment these hydrophobic regions tend to associate, and so the mutant form of the protein tends to aggregate in cells. - In the lower portion of the figure, the misfolded form of the protein can actually associate with the normal form of the protein and induce the conversion of the normal form into the misfolded form. This induced conversion is associated with a number of prion based diseases. In these prion diseases the misfolded form of the protein represents an infectious agent, which when ingested by an animal can interact with the animals normal PrPC protein and induce the conversion and subsequent aggregation of the normal protein. These aggregates are particularly effective at disrupting nerve cell function, leading to neurological and muscular failure and eventual death. Diseases like this in humans is called Creutzfeldt-Jakob Disease, they are also subject to kuru which may result from cannibalism. Scrapie is the version in sheep, and mad cow the version in cows. Although not involving prions and acting in an infectious manner, protein misfolding and aggregation play a critical role in a number of neurodegenerative diseases including Alzheimer's disease.

Proteins provide membrane-specific functions

- Proteins are the second major component of cell membranes, proteins alone cannot form a bilayer, but these macromolecules can associate with phospholipids in a variety of ways, most commonly either by passing through and being dissolved within the bilayer or by associating with phospholipids or other proteins on one face or the other - Membrane proteins provide membrane specific functions, meaning that the specific proteins found on the plasma membrane are critical for the function of the plasma membrane, while the specific proteins found on the surface of the nucleus are critical for the function of the nuclear membrane

Research on cells and tissues

- Research can be divided into 2 broad approaches: 1. Basic research asks "how do cells work?" •Most discoveries that result from basic research can be extrapolated to many types of cells and organisms 2. Applied research uses basic knowledge to "improve life" in direct or indirect ways •Life often means human lives, and classic examples include the understanding and treatment of human diseases and improvement of agricultural or industrial processes that in turn improve the human condition - Driven by: -Human health concerns (NIH, pharmaceutical industry, foundations) -Economic gain (agricultural and pharmaceutical industries, USDA, NSF) -Quest for knowledge (foundations) -Human health concerns and economic gain are two of the main drivers for funding research on cells -Research in this field is expensive and the money invested in this research must yield results that warrant the investment -In the area of human health funding for research is most commonly provided at the federal level by the national institutes of health, generally known as the NIH, funding also comes from the pharmaceutical industry and from various foundations such as the American cancer society or the muscular dystrophy association. The majority of this funding supports basic research, even that provided by companies looking to develop a product like a drug or diagnostic tool because it is essential to understand how cells and tissues normally work, so that one can learn how to fix an abnormal situation. -Economic gain may overlap with human health concerns with companies looking to develop a drug, but research for economic gain may also involve funding to support research on improving crops or domestic animals or developing new biofuel, all of which benefit humans indirectly in which the developer hopes to make a profit. Research in this area is commonly funded by the agricultural and pharmaceutical industries, but some federal funding comes from the USDA (US department of agriculture) and the NSF (National Science Foundation). Much lower on the priority for funding is the quest for knowledge, that is research that simply seeks to add to our knowledge base without any particular expected profit. Some of this research still goes on , but the money available is much less compared to the other two areas and typically comes from various private or public foundations

Scanning electron microscopy provides 3-D images of cell structures

- Scanning electron microscopy collects electrons that are scattered by the specimen. These scattered electrons can be used to produce a 3D image of cell structures - Right FIG. shows two views of the structure known as a stereocilia which is present on hair cells in the inner ear and is critical for our sense of hearing. The Right most image was generated with light microscopy, and the larger image generated with a scanning electron microscope, not only can we see much more detail, we also get a much better 3D understanding of the structure

Some transporters work by passive transport

- Such transport is often termed facilitated diffusion - Like all channel proteins, some transporters also function by passive transport - Passive transport in the case of transporters is often referred to as facilitated diffusion - In the example here, the solute molecule, glucose, id higher in concentration outside of the cell than inside the cell, thus glucose will tend to bind on the extracellular side in a specific binding site that triggers a conformational change and we have a ping pong action so that the molecule of glucose is released to the inside of the cell. This is passive transport, the diffusion is being facilitated by the transporter and the solute is moving down its concentration gradient. After the solute is released to the inside the transporter returns back to its initial state and is ready to pick up another glucose to be carried across the membrane. In this particular situation if for some reason there was suddenly a higher concentration of glucose on the inside of the cell (say we injected glucose into the cell) then at least for a period of time the glucose would actually move out of the cell - The glucose transporter described in this fig is one of the best understood transporters that works by facilitated diffusion, but many transporters for other cargo work the same way

The transmembrane (TM) domain

- TM domain = 20-25 mostly hydrophobic amino acids that form α helix - Most transmembrane proteins contain a so called transmembrane domain which consists of 20-25 mostly hydrophobic amino acids that form an alpha-helix. Some proteins only have one such transmembrane domain and are termed single-pass proteins, while other proteins may contain several transmembrane domains and are refereed to as multi-pass proteins - In the diagram at left we see a transmembrane domain passing through the hydrophobic region of the lipid bilayer, the hydrophobic sidechains extend outward from the alpha-helix and are able to associate with the hydrophobic core of the lipid bilayer, thus the protein passes through without creating any energetic problems in terms of membrane structure and function. In the right hand figure we are looking at an analysis of the relative hydrophobicity of the amino acid sequence of two different proteins, glycophorin and bacterial arabidopsin - In panel a we se that glycophorin contains a single stretch of predominantly hydrophobic amino acids which is highlighted in dark green, thus just by looking at the amino acid sequence of glycophorin we can predict that this protein is a single pass transmembrane protein - In comparison we can see in panel B that bacterial arabidopsin contains 7 predominantly hydrophobic stretches, which is consistent with this protein passing through the membrane 7 times, making it a multi path protein - Even without knowing anything about the location or function of the protein, it is possible based on analysis of amino acid sequence to predict whether a given protein has a transmembrane domain and how many times is passes through the membrane

The Na+K+ ATPase creates and maintains Na+ and K+ gradients across the PM

- The Na+K+ ATPase is electrogenic: 3 + out, 2 + in = 1 net + out - The Na+K+ ATPase creates and maintains Na+ and K+ gradients across the PM - Here is a reasonably accurate situation for a generic animal cell: the sodium concentration is roughly 15x higher outside the cell than inside and the potassium concentration is roughly 30x inside the cell than outside the cell, chloride is significantly higher outside the cell than inside. In addition to generating the sodium and potassium gradience across the plasma membrane, the Na+K+ ATPase also helps to create a charge difference across the membrane, that is because 3 positive charges are exported while only two positive charges are imported, so there is a net movement of one positive charge inside to outside for each ATP molecule to hydrolyze. In this way the action of the Na+K+ ATPase is elctrogenic means it acts to create an electrical difference across the membrane

Phospholipids spontaneously assemble into bilayer when in aqueous environment

- The amphipathic property of phospholipids is critical to the function of these molecules, specifically when phospholipids are placed in aqueous environments these molecules will spontaneously assemble into a variety structures. The most significant such structure for cells is the lipid bilayer which is the foundation of cell membranes. The bilayer is held together entirely by noncovalent interactions in which the hydrocarbon tails associate through hydrophobic interactions and the hydrophilic heads associate with water molecules on one side of the bilayer or the other - The bilayer has two monolayers or faces, that interact with different chemical environments, one outside of the bilayer and one inside the bilayer

Cells have a membrane potential across the plasma membrane

- The cell interior is negatively charged relative to the cell exterior - The difference can be measured as a voltage across the PM (-20 to -200 mV)) - Results in large part from combination of: • fixed anions(Organic compounds, e.g., RNA, PS) • pull cations, repel anions •electrogenic pumping by Na+K+ ATPase - - Membrane potential: there can be a charge difference across the plasma membrane. In this context the cell interior is negatively charged relative to the cell exterior. In fact, the difference in charge across the membrane could be measured as a voltage, which typically measures anywhere from -20 - -200 millivolts. The negative value indicates that the inside of the membrane is negative relative to the outside. The membrane potential is the result of several different factors, but results in large part from a group of organic compounds termed fixed anions. Examples include the negatively charged nucleic acids RNA and DNA as well as phosphatidylserine, which as you recall carries a negative charge and is concentrated on the interface of the plasma membrane. This anions are fixed in the sense that they cannot move across the plasma membrane and thus are fixed inside the cell. The fixed anions attract small cations into the cell, but repel small anions from entering the cell. Another major factor in the generation of membrane potential is the electrogenic pumping from the Na+K+ ATPase, remember this pumps one net positive charge out for each ATP hydrolyzed. Overall the membrane potential of a given cell results from a complex interplay of factors, but the most important are fixed organic ions and the mobile small ions (sodium, potassium, and chloride) the sodium and potassium gradients are largely to due the action of the Na+K+ ATPase, but the chloride gradient may seem unexpected given this ion carries a negative charge. One might expect the chloride will be higher in concentration inside the cell, that is the negative side of the membrane, but notice the opposite is true. This goes back to the issue with anions, the substantial negative charge provided by these organic compounds act to repel chloride and thus the chloride gradient is maintained by electrical rather than chemical forces

Once channel gate opens , ion movement depends on electrochemical gradient

- The movement of ions through open channel protein depends not only on the chemical gradient across the membrane, but also on any electrical gradient across that membrane - In the panel on the left we have a situation in which there is a chemical gradient, but no charge difference across this particular membrane. No channel protein is shown in the diagram, but lets assume one is present, if such a channel protein was to open, the positive ions would move down their concentration gradient from outside the cell to inside the cell - If we look at the situation in the middle, a chemical gradient also exists and is higher in concentration outside the cell than inside the cell. In addition there is an electrical difference across the membrane such that the inside of the cell is negative relative to the outside. In this circumstance not only would positively charged ions move down their chemical concentration gradient but the electrical attraction for the positively charged ions will be such that the negatively charged inside would also act to pull those positively charged ions through. Notice the green arrow here is much thicker than the one to the left, indicating that there is a much stronger pull in this particular case. - The opposite is true if the charge gradient is reversed. In the right hand panel the gradient is such so that there is more ions outside the cell than inside, but the charge difference is such so that it is positive inside relative to negative outside. Thus the positive charge inside acts as a repelling force on the movement of positive ions down there chemical concentration gradient. While there may be some movement of ions into the cell, in general it is not going to be as substantial as either of the other two examples - Movement for charge molecules not only depends on the concentration gradient of the molecule in question, but also on the existing electrical gradient - Note that the electrical gradient across a membrane is also referred to as a membrane potential because the gradient represents a form of potential energy

Cholesterol is abundant in eukaryotic membranes

- The steroid ring structure enhances the barrier properties of membranes - Cholesterol is an abundant molecule in eukaryotic membranes, they even approximate the number of phospholipids in some membranes. - Cholesterol is an amphipathic molecule with a polar head group and a single nonpolar hydrocarbon tail - The head is connected to the tail through a rigid steroid ring structure, which when the molecule is inserted into a phospholipid bilayer, acts to enhance the barrier properties of that bilayer - Unlike phospholipids, cholesterol is unable by itself to form a bilayer structure, and is therefore generally considered an additive rather than a fundamental component of eukaryotic cell membranes

The 20 Amino Acids

- There are 20 types of amino acids that can be broadly classified by whether the R group is hydrophobic or hydrophilic - 10/20 are hydrophobic and are shown in the green Fig. - Of the 10 hydrophilic amino acids, 5 are polar, but carry no charge on the sidechain, these are shown in yellow. The 3 highlighted in red have positively charged or basic sidechains. The 2 in blue have a negatively charged or acidic sidechain - Each amino acid is also identified by a 3 letter abbreviation, and a single letter abbreviation which are shown in the Fig.

Amino acids assemble into a linear polymer via peptide bond formation.

- This Fig. depicts a formation of the peptide bond between the amino acids, glycine and alanine. Peptide bond formation is an example of a condensation reaction that generates a molecule of water. The red circles indicate an oxygen atom and two hydrogen atoms that will form the water molecule. The resulting covalent peptide bond is formed between the carboxylic acid group of glycine and the amino group of alanine. This creates a dipeptide, a structure consisting of two amino acids - On the bottom left, the amino or N-terminus the glycine is exposed, in this end, the peptide is referred to as the amino terminus - On the bottom right, the carboxylic acid of alanine is exposed, and this is referred to as the carboxy end of C-terminus - During protein synthesis, a new incoming amino acid will assemble onto the righthand side of the dipeptide, thus the direction of synthesis is from left to right or from amino terminus to carboxy terminus. The resulting polymer which may consist of tens to hundreds of amino acids is referred to as a polypeptide, only after a polypeptide chain is folded correctly, do we typically refer to the macromolecule as a protein

Different proteins have different shapes and different functions.

- Types of proteins • Enzymes • Structural • Transport • Motor • Storage • Signal • Gene regulatory - Current estimates but the number of protein encoding genes in the human genome at about 20,000, all though not all are expressed in any given human cell, the number of different proteins capable of being produced exceeds 20,000 due to post translational processes such as alternative splicing - Proteins can be categorized in many ways, this slide lists some of the types of proteins based on function - As mentioned in previous lectures, the shape or structure of a cellular molecule is correlated with function, and this is certainly true for proteins

Carbohydrates provide protection & facilitate cell-cell recognition

- While phospholipids generate the fundamental structure of cell membranes, and proteins provide membrane specific functions, carbohydrates also play several important roles, specifically on the outer place of the plasma membrane. Here, carbohydrates help to protect the outer surface of the cell and facilitate cell-cell recognition, that is the ability of cells to recognize and interact with other cells

The four main phospholipids that form cell membranes

- While there are a variety of phospholipids present in cell membranes, most membranes are comprised of the four common phospholipids; PE. PC, PS, SM - phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and Sphingomyelin. The primary difference between these molecules is the chemical composition of each head group, and while all have polar head groups, one phosphatidylserine has a net negative charge

Eukaryotes include__________________________, and what are some common features

- animals, plants, and fungi, there is also a wide range of single cell eukaryotes - contain a nucleus and other membrane bound compartments (often referred to as organelles) - some posses exterior cell wall (plants and fungi) others (animals) do not - some are unicellular (yeast and various amoeba), others are multicellular (plants and animals) - generally are bigger than prokaryotic cells - some have motile appendages that allow them to swim through liquid

cells vary enormously in __________ and ______________- despite phenotypic and functional differences

- appearance and function - all living cells exhibit the same fundamental chemistry - all present day cells have evolved from the same ancestral cell (related)

Eukaryotic cells contain a dynamic system of protein filaments that form the _________________

- cytoskeleton - microtubules are involved in vesicle transport, organelle positioning, mitosis, and cell swimming - active filaments are involved in the generation of cell shape, cell contraction, and cell crawling - intermediate filaments help to resist mechanical stress and support the nuclear membrane - with an exception to certain intermediate filaments inside the nucleus, all 3 cytoskeleton systems are located in the cytosol - Fig. shows an image of an animal cell in which a technique called fluorescent microscopy has been used to reveal the location of two cytoskeleton filament systems; microtubules, and active filaments as well as the location of the nucleus. Microtubules are labeled in green, active filaments are labeled in red, and the nucleus in blue.

common features of archaea and bacteria

- no nucleus or membrane bound internal compartments surrounded by membranes- the absence of a nucleus lead to such cells being referred to as prokaryotes, which is derived form the Greek "before not, or before kernel" which is before nucleus - an exterior cell wall just outside the plasma membrane which supports and protects the cell - generally unicellular - generally smaller than eukaryotic cells - some have motile appendages (like a flagella that helps cells swim in liquid)

~ 70% is water, so ~30% of the remaining cell mass is represented mostly by _________________

- organic molecules based on carbon - each of the major classes of cellular chemicals are associated with specific cell processes - 4% of the cell mass are Inorganic ions and small molecules which are used in a cell to signal the control of the pH of the cell interior and for energy sources like ATP. - 2% of the cell mass is phospholipids, they minor in abundance but are critical for the structure and function of cell membranes.Some related lipids, and especially fats serve as important energy sources - overall the most abundant cellular molecules after water are the macromolecules. - DNA comprises about 1% of cell mass, while RNA accounts for 6 times more- these are critical for the storage and expression of genetic information - proteins comprise the largest amount of cell mass (15%) other than water. They most commonly function as enzymes or provide structural support. - 2% by polysaccharides which function to store energy and may also be found on the outer surface of cells where they function in cell protection ans regulate cell to cell interactions

Phospholipid bilayers exhibit selective permeability

- slower than nonpolar - Different molecules inside vs. Outside - The bilayer assembled from phospholipids creates a selective barrier to the movement of molecules from one side of the bilayer to the other, that is through the hydrophobic core. This barrier is selective, in that some molecules diffuse across the bilayer, while others cannot - Small Hydrophobic molecules and readily diffuse to the bilayer, while small uncharged polar (hydrophilic) molecules like water may do so, but at a slower rate. In comparison, larger uncharged polar molecules like glucose or amino acids almost never directly defuse to the bilayer and charged molecules, even small ions are not able to penetrate through the hydrophobic core of the bilayer at all - The Right Fig shows how the selectivity of the membrane barrier allows for the maintenance of chemical differences across the plasma membrane and across internal membranes. These differing chemistries are critical for the functions of all cellular compartments, including the cytosol

What did the understanding of cells begin with

- the invention of the microscope in the 1600's - allowed scientists of this period (Robert Hooke, Anton Van Leeuwenhoek) to visualize cells for the first time. Took roughly another 200 years for the guiding principle of cell biology, the cell theory to mature to its current form - the cell theory and theory of evolution continue to guide our overall understanding of biology

Most abundant cellular molecule is _________, thus the cell interior is an __________ based environment

- water, aqueous/water - water comprises ≥ 70% of a cells mass - a cell is an aqueous environment and lives in an aqueous environment - Understanding how a cellular molecule functions depends on understanding how that molecule interacts with water. Cell molecules may be * Hydrophilic: able to readily interact with water (polar) * Hydrophobic: will cluster together in water based or aqueous environments * Amphipathic: both, some molecules have hydrophobic and hydrophilic regions, function of these molecules is critically dependent on whether they are able or unable to interact with water molecules

Light microscopy allows observation of living cells.

-The most common type of microscope is the light microscope, in which the visible light is directed through a specimen, for example cells, and the resulting image depends on the ability of the cells to absorb light to varying degrees. - Animal cells in the left fig have little inherent contrast because they don't contain many molecules that absorb light, this also shows the abundancy of water in the cells, because water is abundant and does not absorb light, there is not a lot of contrast -Plant cells in the right fig. there are structure like chloroplast that are able to absorb light and thereby show a contrast allowing us to easily see their organelles -Certain types of light microscopy can assist in generating contrast, this is generally done by manipulating the phase where polarization of light is in the microscope -In the middle left fig. the same cell in the top panel is observed by phase contrast microscopy. In the bottom panel the cells are viewed by differential interference contrast microscopy. Both techniques use optical techniques to generate contrast, and thus reveal features not seen with the simple microscope -A significant benefit of light microscopy is the ability to observe living cells, so we can follow the behavior of cells over time. -Both the animal and plant cells in the Fig. were observed while alive

3 tenants or principles of cell biology

1. All organisms are composed of cells - in the case of unicellular organisms like bacteria or yeast, the cell is the organism. In the case of multicellular organisms, like us, the organism may be composed of trillions of cells 2. Cells are minimum functional units of organisms - cells are the "essence of life" - lowest entities that are considered alive 3. all cells arise from pre-existing cells by the process of cell division - no spontaneous generation of cells

Lesson 1

Cell structure and composition

Microscopy is the founding technique of cell biology

Microscopy provides: 1. Magnification => Allows enlargement of the specimens image. This is the feature most people associate with the microscope, and while important, magnification alone is not sufficient to make a microscope useful 2. Contrast => Anility to define the specimen from the background, or different parts of the specimen from each other 3. Resolution => The ability to discriminates two objects from one object. The closer two objects are to each other and can still be recognized as two separate objects, the better the resolution -Invention if the microscope directly lead to the discovery of the cell, therefor microscopy is considered the founding technique of cell biology -It started off as an observational or descriptive technique. While still prominent in that way it has evolved to become an experimental technique in which cells can be manipulated by or through the microscope, and the result of such experiments may be recorded -Whether descriptive or experimental, there are 3 critical features that any microscope must provide to be useful to the researcher -Word image is emphasized because the microscope only generates an image of the specimen, and image quality depends on many factors. It is important to keep in mind that no image is perfect and the relative accuracy can effect what one sees or thinks they see -Magnification, contrast, and resolution, are to some degree linked to each other, but all things being equal scientists want the best resolution possible

lesson 3

Proteins - they are responsible for most of the day to day activities within the cell

How then, do noncovalent bonds play such an important role for cellular molecules and for life itself?

The answer involves the ability of noncovalent bonds to both form and break relatively easily, which allows for critical but temporary interactions to both occur and terminate on an as needed basis. While a single noncovalent bond is weaker than a single covalent bond, macromolecular interactions generally depend on many noncovalent bonds, which provides the appropriate strength to the interaction, as well as the specificity for the interaction, as the right bonds must be formed at the right locations to allow two macromolecules to interact

Genetic information is expressed according to ________________

The central Dogma - DNA, RNA & proteins are directional polymers, in which the specific unit of subunits contains the information stored in that particular macromolecule => info stored in the sequence of subunits -A macromolecule, such as a protein or a given RNA molecule, performs a specific function in the cell -Genetic information is stored in the form of double stranded DNA. Each strand is assembled from four types of nucleotide subunits, usually referred to by their single letter abbreviations, A, T, C, and G. information contained in one of the DNA strands is converted to a single stranded intermediate form, RNA by the process of RNA synthesis or transcription. Like DNA, RNA is also assembled from four types of nucleotide subunits, A, U, C, and G. information in RNA specifically mRNA is converted to a functional form, a protein, which is a polymer assembled from 20 different types of amino acid subunits, this process is referred to as protein synthesis or translation. The expression of genetic information to functional product through transcription, followed by translation is termed the central dogma of biology -While expression of genetic information is considered to occur according to the central dogma, there are always exceptions, for example, there are several classes of RNA such as transferRNA and ribosomalRNA which act as functional products in their own right and never serve as a sequence template for the assembly of a protein - Genes are discrete DNA sequences that code for a functional product - Only a portion of any cell's genes are expressed - Which genes are expressed can vary with time and situation - Of course a given cells DNA is not completely and totally converted into RNA, DNA sequences that code for a functional product are termed genes and not all the cell's genes are expressed at a given time. Which genes are expressed varies by organism, cell type, and environmental conditions. Ongoing efforts to identify genes and important DNA sequences through genome sequencing have yielded a wealth of useful information. However, trying to understand which genes are expressed at any give time and why is a much more complicated, though critically important task

Macromolecule assembly requires __________

energy - The formation of covalent bonds during replication, transcription, and translation proceeds by the condensation reaction in which a molecule of water is produced - this FIG. the condensation reaction leads to a covalent bond between subunit A and subunit B. Such reactions are energetically unfavorable, and thus the assembly of a macromolecule requires and energy input, usually in the form of ATP hydrolysis, in order for the condensation reaction to proceed. In comparison, breakage of the covalent bond between macromolecule subunits occurs via hydrolysis and is energetically favorable, though it still requires an enzyme to lower the activation energy of the reaction in order for the reaction to occur.

Cells constantly obtain and expend ______________ maintain structure and function

energy - The assembly of a macromolecule, is just one example of a cellular process that requires energy. To maintain their overall structure and function, that is to be alive, cells must constantly obtain and expend energy. Much like the room in the figure at left, the universe follows the second law of thermodynamics; disorder is continually increasing. Energy is required by living organisms to offset the increasing disorder or entropy, just as energy is required to maintain the room in an organized state. Most organisms obtain such energy from the food molecules they ingest, as shown in the FIG. on right, by degrading such food molecules, cells can obtain energy ultimately in the form of ATP as well as the building blocks or subunits needed to assemble more macromolecules through various metabolic pathways. The degradation of food molecules through catabolic reactions is not 100% efficient and some of the energy stored in the food molecules is given off as heat to the surrounding environment, which increases the disorder of the environment, even as the cell is maintaining an ordered state

macromolecules interact with other molecules via _____________________ bonds

noncovalent - although macromolecules are formed through covalent connections between subunits, the ability of a given macromolecule to carry out the specific function in the cell depends on its ability to interact with other cell molecules, both small and large. Such intramolecular interactions (the interactions between two different molecules) largely depends on noncovalent bonds rather than covalent bonds - once macromolecules are assembled through the formation of covalent bonds the resulting macromolecules may assemble to create more complex cellular structures, such as a ribosome, which consists of many different protein molecules, and several different RNA molecules, all held together by noncovalent interactions. - A covalent bond is stronger than a noncovalent bond in aqueous environments such as the cell

bacteria and archaea are ________________

prokaryotes

Lesson 2

research methods

Energy flows from ____________ to _________________ to _______________-

sun, organic molecules, ATP - The sun is the ultimate source of energy for life on earth: plants, algae, and certain bacteria have evolved to carry out the process of photosynthesis, thereby converting the energy from sunlight into sugars and other organic molecules. As a byproduct of these photosynthetic reactions, molecular oxygen is also produced. The organic molecules actually produced by photosynthesis serve as an energy source or food that can be utilized by most other living organisms. Such organisms carry out the process of cellular respiration in which organic molecules are oxidized to carbon dioxide and reactions that utilize molecular oxygen to convert the energy stored in food into chemical bond energy, mostly in the for of ATP. Photosynthesis and cellular respiration are complementary processes that change energy from one form to another and interconnect all life on earth

The function of the macromolecule depends on the subunit _____________ and ______________

type and sequence - simple sugars, or monosaccharides such as glucose or fructose are the building blocks of polysaccharides. In comparison, amino acid subunits are assembled to make the nucleic acids, DNA, and RNA. The various subunits may form shorter chains that also have important cellular functions. These shorter chains of monosaccharides are termed oligosaccharides, and short chains of nucleotides are similarly called oligonucleotides, whereas short chains of amino acids are termed peptides

Proteins consist of one or more domains .

• A domain is a module of tertiary structure that has a specific function - Most proteins consist of one or more structural elements termed domains. Domains are modules of tertiary structures that have a specific function, many proteins consist of multiple domains. For example, the protein on the right consists of two different domains, each of which presumably has some specific function that is important for the overall activity of this protein. The given protein domain is constructed of various secondary structure elements connected by flexible runs of the polypeptide, termed bloops, the 3D structure of each domain is critical in turn with a specific function of that domain within the overall protein

Systems approach seeks to understand cells holistically

• Cell is more complex than the sum of its parts • Focuses on networks and interactions within and between cells • Relies heavily on computational and mathematical modeling • Make predictions that can be tested experimentally - Although the reductionist approach has been and remains the primary guiding philosophy used in research on cells, more recently some scientists have pursued a different perspective known as the systems approach. - In this approach scientists seek to understand cells holistically, that is whole or completely, in this view the cell is more complex than just a sum of its parts. The systems approach works on networks and interactions both within cells and between cells. It relies heavily on computational and mathematical modeling to handle the number and complexity of interactions between cellular molecules. This modeling allows for predictions to be made that can be tested experimentally. Ideally the experimental results can be used to further refine and improve the model, with the ultimate goal being to understand the cell in total

Electron microscopy uses electrons to illuminate the specimen

• EM provides better resolution but can't observe living cells and procedures may generate artifacts - In a schematic of the light microscope as depicted in the Left FIG, the specimen is illuminated by visible light focused by various lenses, in comparison the electron microscope uses electrons to illuminate the specimen - As seen in the Right FIG, the beam of electrons is focused on the specimen by magnets, rather than by the glass lenses used in a light microscope. The resulting image is then viewed on a screen or piece of film. - while electron microscopy provides superior resolution, this benefit comes with a trade off. Electrons are scattered by air which means the specimen must be placed in a vacuum so the electrons can illuminate the specimen, thus we cannot look at living cells in an electron microscope. Further the specimen must often be fixed, sliced into sections, and stained with electron dense material in order to facilitate imaging. These manipulations can induce artifacts that can make it difficult to be sure whether the image obtained is an accurate representation of the specimen. As with a similar approach for light microscopy, one must balance the pros and cons of the approach when using electron microscopy - This method is termed transmission electron microscopy because the electrons are directed onto the specimen and some pass through to the protection screen of piece of film.

Resolving Power: Light vs. Electron Microscopy

• Electron microscopy provides better resolution than light microscopy - Just as some scientists worked on techniques to improve the contrast of specimens for microscopy, others worked on ways to improve resolution. These efforts lead to the development of the electron microscope, which quickly became an important tool for research on cells and tissues. - The primary reason a scientist would want to use an electron microscope is because these instruments provide better resolution than the light microscope. This results from the fact that electrons have a shorter wavelength than visible light - In the FIG of a scale in the Left, we see that resolution range of the unaided eye, the light microscope, and the electron microscope, as well as the various sizes of various biological specimens. The distances indicate how close objects could be while still being able to distinguish them as two separate objects. The smaller the distance, the better the resolution. The lower limit of resolution for the light microscope is about 200nm, two objects that are closer than that will appear as one object in a light microscope, but an electron microscope would be capable of resolving them as two separate objects. - In the right FIG, we see a few different examples of images produced with an electron microscope. Panels A and B show the section of a liver cell at two different magnifications. Not only can we easily resolve individual mitochondria, it is also possible to see individual ribosomes, which are well below the resolution limit of the light microscope. We can even visualize a single DNA molecule with electron microscopy, as shown in panel C

A protein's primary structure is its amino acid sequence.

• Generated by covalent peptide bonds between adjacent amino acid sub units - Proteins are considered to exhibit 4 levels of structure 1. primary structure: simply the amino acid sequence of the polypeptide chain, in the Fig. we see a portion of 189 amino acid protein. As already described, the amino acids in the polymer chain are liked together by covalent peptide bonds during the process of translation, although essential for the eventual formation of a functional protein, the unfolded primary structure alone is not functional. For example, an enzyme in its unfolded primary structure will not be able to catalyze the appropriate chemical reaction 2. secondary structure 3. tertiary 4. quaternary

Secondary structure is formed by H bonds between nearby amino acid subunits

• H bond created between amino group hydrogen and carboxyl group oxygen • Often can predict from analysis of amino acid sequence 1. primary structure 2. secondary structure: typically generated by bonds formed nearby (close to each other in the amino acid sequence of the protein) amino acids. One of the two common types of secondary structure is the alpha-helix shown in the Left Fig. This structural element is generated by the formation of hydrogen bonds between an amino hydrogen and are carboxylic acid oxygen for amino acid separated by 3 intervening amino acids. This bond is therefor formed between elements of peptide bonds, and does not involve interactions of side chains 3. tertiary 4. Quaternary - Left fig shows three different depictions of and alpha-helix, including the standard ribbon cartoon shown in panel C. - The other major example of a proteins secondary structure is the beta-sheath. With the alpha-helix, hydrogen bonds form between the amino group hydrogen and the carboxyl group oxygen. Unlike the alpha-helix, these bonds form between short stretches of amino acids that run past each other in a side by side fashion creating a flattened sheath like structure - Right Fig. shows 3 depictions of a beta-sheath with panel F depicting the commonly used ribbon diagram - The arrows indicate the direction of the polypeptide running from n-terminus to c-terminus. - Because the amino acids that create secondary structures are relatively close to each other in the primary structure of the protein, scientists can often use computational methods to predict the location of alpha-helix's and beta-sheaths just by analyzing the proteins in an amino acid sequence

Cells can be grown in culture .

• Many examples of unicellular organisms and of cells from multicellular organisms Cell culture requires appropriate liquid media and appropriate dish or flask -Many cells can be produced through artificial cells in the laboratory, could be cells form a uni- or multi- cellular organism. - Cell culture requires an appropriate liquid media with nutrients and chemical conditions that will support cell growth and survival. - Cells grown in culture are usually placed in a dish or flask, the dish supports cell growth on the surface, while a flask will e used for cells that grow in liquid. - Fig. shows different types of animal cells growing on the surface of a culture dish, the use of cultured cells provides a number of advantages to researchers, including the opportunity to conduct controlled experiments in a relatively simple environment in which single cells can be readily observed - While one hopes the cultured cells will behave as they do in the organism, researchers must always keep in mind that the environment in the dish or flask is not the same as the inside of an organism, and results must be interpreted with this cavoite is mind

Stains provide contrast of specific cell components

• Often must section and fix specimen to allow stain to penetrate cells • Can't observe living cells and procedures may generate artifacts. - As the field of microscopy continues to develop, scientists search for ways to improve contrast and also visualize certain parts of cells, or even certain molecules within cells - The two Figs. Show sections of images that have been stained to enhance contrast -Left Fig. is a section of plant root tissue which has had nuclei stained red, and cell wall has been stained blue - Eight fig shows section of kitty tissue with nuclei stained red and the extracellular matrix has been stained purple - It turns out that many of the stains now used to generate contrast for microscopy were originally developed as textile dyes in the 1800's. The specimens in the image were both sectioned into thin slices and fixed with formaldehyde. This procedure is necessary to both reduce the specimen thickness and to allow the stain to penetrate the cells - The consequence is that we cannot observe living cells with this approach, and the fixation procedures may generate artifacts that could alter the specimen, thus there are trade offs one must balance when deciding how best to image samples of cells and tissues

Protein synthesis is not sufficient to produce a functional protein

• Polypeptide must fold correctly to form 3-D structure • Proteins with similar sequence and structure are members of a protein family - Assembly of amino acids into a polypeptide chain is necessary but not sufficient to produce a functional protein - A linear polypeptide chain is not functional, the only way it becomes functional, is if the polypeptide chain that is the amino acid sequence to fold correctly to form a 3D structure. - In this Fig we see the folded 3D structures of 2 enzymes The red region indicates the active cell of these enzymes, the only way this active site can be formed is for the polypeptide chain to fold to create the correct structure and 3D space - This fig should also serve as a reminder that proteins may be similar to other proteins, both in amino acid sequence and 3D structure, as is the case for the enzymes pictured. - Proteins with similar sequence and structure are considered members of the same protein family, which are ultimately related to each other because they are coded for by members of the same gene family

Animal cell culture

• Primary cells divide 50-100 times then become senescent and eventually die • grow until confluent then subculture • Mortal = limited reproductive capacity - animal cells can be cultured from tissue obtained either following sacrifice of the animal or a nonlethal biopsy of a small amount of tissue. In either case, the tissue sample is subjected to physical, chemical, or enzymatic means to disrupt the tissue structure and release individual cells. - In the Left FIG, a homogenizer which is similar to a mortar and pestle, is used to break up the tissue and release individual cells. These disrupted cells are then added to a dish containing appropriate growth media, a solution of nutrience ions, and other molecules that promote cell survival and reproduction. The cells attach to the surface and proliferate until the bottom of the dish is covered, at which point the cells are said to be confluent, or have reached a confluence. A small group of cells can then be removed from this dish and placed in another dish where they will reproduce until the cells again become confluent. This process can be continued over several so called passages, but the cells will eventually die. The cells obtained from an organism in this way, as well as their descendants are called primary cells. - If we plot cell number as a function of time, primary cells behave as shown in the Right FIG., the cells reproduce and increase in number for a period of time, which may involve passage to several sequential new culture dishes, eventually reproduction stops and the cells enter a state termed senescence, where they have lost the ability to reproduce. Cells that become senescent may live for various periods of time, but will eventually die - Primary cells are referred to as mortal, which means they have a limited reproductive capacity, typically primary cells can reproduce for 50-100 generations

Fluorescent microscopy allows visualization of specific cellular molecules.

• Provides a high level of contrast and can be used for living or fixed cells . - It is possible to visualize different organelles and the cytoskeleton in living organisms as well as fixed cells by a special type of light microscopy called fluorescent microscopy - In this technique fluorescent molecules are targeted to specific cell components such as organelles or a certain type of protein or even a certain DNA sequence - The location of the fluorescence reveals the location if the cellular molecule and any structures the molecule may be associated with - The FIG shows the network structure of the endoplasmic reticulum on the LEFT and the cytoskeleton and nucleus at RIGHT - Provides a high level of contrast because the molecule of interest is very bright on a dark background

Tertiary structure represents a properly folded 3-dimensional polypeptide

• Tertiary structure is generated by interactions between amino acids far apart in the primary structure - May involve: • H bonds . • Ionic interactions . • covalent (disulfide) bonds • Hydrophobic interactions . - Not yet able to predict from analysis of amino acid sequence 1. Primary 2. Secondary 3. Tertiary: for many proteins the folding of the polypeptide chain to form tertiary structures is sufficient to produce a functional molecule. Unlike secondary structure, tertiary is generated by interactions between amino acids far apart from each other in the primary sequence of the polypeptide. These interactions may involve hydrogen bonds either between atoms of a peptide bond and a sidechain or between atoms of two different sidechains, as highlighted in yellow and blue of the Fig. Other interactions that contribute to tertiary structure include ionic interactions between acidic and basic sidechains, the formation of covalent disulfide bonds between two cystine sidechains and hydrophobic interactions between nonpolar sidechains. Because of the complexities involved in predicting interactions between distant regions of a polypeptide, it is not yet possible to routinely predict a polypeptide's tertiary structure by analysis of its amino acid sequence. Much research effort and computational power is being brought to bear this issue that is may be possible eventually to go directly from knowledge of an amino acid sequence to knowledge of that proteins tertiary structure 4. Quaternary

Reductionist approach seeks to understand cells in terms of molecules

• identify every molecule in "the Cell" -> vertebrates have about 200 different types of cells • Determine function of each molecule • Determine mechanism by which each molecule functions -Research on cells has historically take a reductionist approach. In this approach scientists wish to understand how the cell's components work in order to understand how the cell itself works. -Scientists look to discover 3 types of information in this approach 1. First they need to identify every type of molecule in "the cell" -"The cell" is in quotes because much of the material in this class is fundamental to all or at least many types of cells, so "The cell" will often generically be used. For example, when a researcher learns about yeast cells many may often be extended to plant and animal cells, and vice versa. Keep in mind, for a multicellular organism, molecules from one type of cell may differ in varying degrees from a molecule from different type of cell. In vertebrates, there are roughly 200 different types of cells 2. They have to determine the cellular function of each type of molecule. -Function at the molecular level is tightly correlated with the structure of a given molecule. The relationship between structure and function is found throughout biology, whether we are talking about molecules, cells or even organisms 3. They need to determine the mechanism by which each cellular molecule preforms its function -Basically the reductionist approach asks 3 simple questions about cellular molecules, who, what, and how