Cell Biology, An Overview Pt 1

The first in a series of notes for Cell Biology. This would prove helpful if you're attending a college level Cell Biology course, as I attend a prestigious university. This should, by no means, be the only resource used to learn about Cell Biology, nor is it fully complete. However, for those who are looking for a brief overview of the topic, this is definitely your read.

Cell Biology

  1. By the use of complementary DNA sequences, you can use Southern Blotting to separate DNA based on size on a gel. Afterwards, the DNA can be transferred to a membrane-paper thing, and then using a radiolabeled probe, you'll see bands where the probe has bound.
  2. Histones can be modified by phosphorylation, acetylation, and methylation. Modification will lead to the activation/deactivation of histones working or not. If you add an acetyls to a group of chromatin, you end up activating it for transcription. Acetylation gives the histone a negative charge, so it will repel the DNA chain and allow it to loosen.
    1. Starting with euchromatin, you can add H3K9 methyl transferase (modifies histone 3 in the 9th position by adding methyl) . HP1 proceeds to bind to the methyl groups – when HP1 gets close to one another, they stick to one another – resulting in your DNA being contracted. Boundary elements separate active euchromatin from the silenced HP1 coated heterochromatin.
  3. Epigenetics are heritable phenotypes with no change in DNA sequence. Stem cells divide to become differentiated cells. Histone changes can be passed on through cell divisions – the differentiated cell's histone code is passed on, not the stem cell's, even though the DNA sequence has not changed.
  4. Proteins have a N-terminus on the Amino End, and a C-terminus on the Carboxyl End. Primary structure of proteins is the amino acid chain that makes it up, secondary is alpha-helix or beta-plated sheets. Hydrogen bonding is what helps to cause the secondary structure to fold and form. A folded protein has hydrophobic groups and hydrophillic groups – folding tries to minimize contact of hydrophobic groups with water.
  1. In the Proteosome degradation sequence, certain enzymes will only interact with another specific enzyme that follows it. E1a → E2a → E3a. Each of these enzyme/proteins in question here can exist in a phosphorylated and a non-phosphorylated protein. Phosphorylation influences the activation of the protein – each protein can be regulated using this method. Ubiquination chains onto the target protein in question, which is then recognized by a proteosome that can recognize these poly-ubiquinine chains and degrades the protein in question.
  2. Carbohydrates are hydrocarbon structures that exist as monosaccharides (sugar). Most often they are used as an energy source like glycogen or starch. It can also exist in cellulose. Even ATP has a carbohydrate as part of it.
  3. Sugars can be conjugated into a side chain by glycosylation. This can result in a complete change of the protein, including hydrophillicity, viscosity change, and even making them resistant to proteolysis. This can lead to complex, branched side chains.
    1. A glycoprotein has a few carbohydrate groups bound on say, the serine group.Proteoglycans are mostly carbohydrates with a protein backbone.
    2. N-linked glycoproteins (linked by the N-terminus) have the same backbone shared. There's a conserved part of the protein. O-linked glycoproteins do not have the shared core part.
    3. There's a lipid in the ER (endoplasmic reticulum) called Dolichol Phosphate. Then some UDPs with a N-acetylglucosamines come here and give it phosphate groups, until it has built a fair amount of N-acetylglucosamines, when the 5-GDP comes with mannose. Then the lipid flips over so it's now facing the cytosol inside the cell, where further mannose are added. The core unit composes of 2 glucosamines and 3 mannoses, so they have to add at least that much.
      1. The protein then comes in after your completed backbone is here, glucose, mannose, and all. Once the protein enters, an enzyme causes the transfer of the glyco-side chain onto the protein..
  4. Fatty acids consist of a glycerol group and 3 fatty acids – the 3 fatty acids determine what physical property, if any, the lipid would have. Fats are most commonly used for storage and energy purposes.
    1. Another compound in this category is the phospholipid, which consists of hydrophobic tail, as well as a head group (serine, etc). The third carbon is attached to the phosphate group. These lipids compose biological membranes.
    2. And yet another compounds in this category is sterols (like cholesterol). They compose of 4 rings and a few side chains. Sterol lipids are important as hormones.
    3. And yet ANOTHER compound is sphingolipids. They look pretty similar to the phospholipids on first glance, but they have a NH group on the tail, as well as sphingosteen group. These are used for signaling communcation, and are also important in membranes (not surprising due to their hydrophobic tail and hydrophillic head).
  5. The Glycolysis and TCA cycle is essential to many of the products involved in cell biology, such as cholesterol and amino acids. Not only does this cycle generate energy, but plenty of important products.
    1. A lot of enzymes in the cell are regulated by allosteric mechanisms (small molecules can bind to proteins of interest to mess with their regulation). For example, the production of Fructose 1,6 – Bisphosphate is regulated by PFK-1, which is further regulated by Citrate and ATP, which tone down the PFK-1, and AMP and Fructose 2,6-Bisphosphate (produced by Insulin) will activate the PFK-1 further. If you don't have enough energy, PFK-1 is active.
    2. In the regulation of cholesterol synthesis, we look at the limiting step for HmG-CoA reductase. When we have normal levels of cholesterol, HmG-CoA will continue to manufacture cholesterol, but when we have elevated levels, cholesterol binds to HmG-CoA which then causes ubiquinine molecules to bind to HmG-CoA and this leads to protease destruction of the HmG-CoA, thusly toning down the expression of this enzyme when cholesterol levels are high.

A re-cap of Techniques (Brief)

  1. Let's talk about microscopy. Resolution is the ability to see two separate closely spaced dots as two dots. Diffraction refers to the behavior of waves (not just light) when it reaches an obstacle. In microscopy, light bends and spreads out, redistributing w/ an obstacle in its way. In this perception, we also have interference, which is what makes the image fuzzy and unclear when we see it. The longer your wavelength is, the more diffraction you'll be seeing. In order to improve our resolution, we can decrease wavelength (like a blue light, which is a low wavelength), or even better, use electrons.
    1. Too bad living things we study in biology don't like electrons – you would have to look at dead samples.
    2. Fortunately, we have PALM, which allows us to look at fluorescence, but only at certain sets of fluorescent proteins separately at different points in time/space. The technique only illuminates a certain part of the sample with light for a very short amount of time, and only certain proteins will fluoresce, not all of them. You can then photobleach, which stops the proteins that fluoresced from doing it again. You can then use computers to calculate where the fluorescent proteins are
      1. The result is different photos of the cell with different fluorescent proteins, and then you combine them altogether for the whole picture – now minus the interference and diffraction since you only see a few proteins a few time.
    3. Another technique, called STED, relies on a limit of which how much you can focus a beam of light. By using Stimulated Emission, you can “artificially” focus – you hit a section with a certain color of light, then hit it with another light color, which limits the size of the effective excited area (smaller area illuminated, more focus).
  2. When you add cholesterol to a phospholipid chain, you change the structure of the phospholipid and change the diffuseability and viscosity and fluidity of the membrane. These additions of proteins allow changes in the lipid bilayer by having the cholesterol change the membrane at these areas. Lipid grafts are an area where there is less fluidity in the membrane – one way to achieve this is by cholesterol.
    1. A technique called FRAP utilizes fluorophores – however, when a fluorophore becomes bleached, from prolonged exposure to excitation, do not light up anymore. Using this idea, you can label proteins on a cell, and then bleach an area using a laser, so if the membrane was immobile, you wouldn't see these proteins move.
  1. Ion channels do not need to generate energy in order to move ions around. Coupled Ion channels will allow between 1 and 2 ions through. A single ion being let through is a uniporter. Two ions with different concentration gradients, such as one with higher concentration outside of the cell and one with higher concentration inside, when the higher concentration moves into the cell, the energy decrease is used to transport the other ion into the cell at the same time, even though it's moving against the concentration gradient. An antiporter has ions moving in opposite directions
    1. This form of transport is called 'secondary active transport', since it's moving ions using energy, even though it's not reliant on ATP. The professor has a slide that discusses these things.
    2. Transporters can distinguish ions. Potassium channels for example use residues of oxygen that look similar to water, which bind to potassium relatively weakly. Now, if a sodium tried this channel, the potassium is a larger ion, so it'd rather bind to the water molecules floating around, because it could only bind to 2 of the 4 residues.
    3. Any time you need to go from a more stable state to a less stable state, you need to expend energy. Because of this, it may be thermodynamically unfavorable for sodium to go in a potassium pore.
  2. In the real world example of cystic fibrosis, there are over 1000 mutations that can cause the disease.Cl- export is required to make mucus less dense – cystic fibrosis in general causes the mucus to clump up and be heavy.
  3. On the basolateral surface of the cell, we have a symporter that imports potassium from the blood, and exports sodium. On the apical surface, which touches the intestinal tract, has a symporter that moves glucose into the cell and sodium into the cell at the same time. This symporter is on the glucose side because food releases the glucose in question. Without proper glucose absorption however, GLUT2 will simply move the glucose out into the bloodstream.
    1. It's important to know that Glucose/Na+ symport causes water absorption into the bloodstream as well. Many diseases will result in dehydration – that's why drinks contain sodium and glucose in order to get water into your bloodstream (the symporter will help move water).
  4. If you wanted to acidify an organelle, it would be necessary to have a proton pump go ahead and move hydrogens into your organelle, but at the same time, move chloride ions in to neutralize any electric potential charge, so you get acidification properly.
    1. In the stomach, you move hydrogen ions into the stomach lumen through a symporter that moves potassium into the cell. Another channel for chloride moves chloride into the stomach from the cell. But how is the homeostasis of the cell maintained if it keeps losing ions? The breakdown of water leads to OH- and H+, but CO2 is transported in by diffusion which will bind to OH- and form HCO3-. HCO3- is antiported with chloride in the bloodstream, and that's how chloride is moved into the cell.
  1. F-Class ATP pumps convert PMF to ATP – they rely on the energy of the proton motive force to generate ATP. Experimental evidence using bacteriorhodopsin (a protein that can convert light energy into a PMF), along with ATP synthase and an artificial membrane, you can look at the ATP produced. Without any light, you don't get any ATP because the bacteriorhodopsin doesn't pump any H+ into the 'cell' in the first place.
    1. The PMF couples electron transfer to ATP synthesis – the electron transport chain sends hydrogens out of the membrane, and the ATP synthases let hydrogen back in, and form ATP. ATP Synthase is conserved throughout species. Without PMF, you have carbon fixation in plants, leading to no life in general.
  2. What are some characteristics and functions of organelles? How are they regulated (A cell may not want to keep producing ATP all the time, which may exhaust another supply)? How did organelles form (biogenesis)? A cell needs to make sure it couples the cell's needs and the cell's own division to the biogenesis of organelles.
    1. One of these important organelles are mitochondria. Mitochondria are very important in free radical breakdown – these atoms that have an odd number of valence that are highly, highly reactive. Normal cell metabolism generates free radicals, which the mitochondria are tasked with breaking down. It also performs the breakdown of energy molecules like carbs and fats, as well as tissue-specific functions such as steroid hormone synthesis and neurotransmitter metabolism, and heme synthesis.
    2. The number of mitochondria in a cell must be regulated. In yeast cells, by mutating what the mitochondria can do, you can get interesting structures. For example, by growing yeast cells at a lower temperature, which is okay, but then moving them to a higher temperature, then the mutation may kick in and you'll see a phenotype be expressed, like the inability to divide
    3. One thing to keep in mind is that mitochondria have their own genome – during division of mitochondria, you need to have it correlate with the cell genome, since only 37 genes for mitochondria are coded by the mitochondrial DNA and the rest is from the cell. Mitochondria numbers are influenced by cell division and cell growth.
      1. The way the cell regulates this is using a protein coded by the cell called Tfam that will assist the mitochondria's replication and division – all thanks to one protein. There are other proteins that also cause fusion, if so desired. By regulating the proteins for fission and fusion, you can drive a mitochondria to one or the other.
  3. Endoplasmic reticulum is an organelle that is mostly membrane, and is continuous with the nuclear envelope. The ER is used for protein synthesis, organelle proteins, and secreted proteins. It's also useful for detox, lipid metabolism, and membrane synthesis. It has some tissue-specific uses, like Ca2+ regulation in Muscle SR.
    1. ER is never synthesized de novo. Lipid addition and membrane extension are how the ER actually extends, along with the fusion of vesicles. ER is inherited through cell division, and is split apart for division. How is it actually divided?
    2. There's two models for the division of ER. During cell division, the nuclear envelope completely breaks down. Now, the question is, it can either form vesicles, with nuclear pore membrane proteins and inner nuclear membrane proteins forming their own individual vesicles, OR the ER network will incorporate the nuclear proteins equally throughout the structure, which is pulled apart upon division.
    3. The Golgi Body is a dynamic structure composed of cisternae. The cis face is closest to the nuclear envelope, whereas the trans face is furthest away. Golgi Body is assembled from ER vesicles
      1. The cisternal maturation model for Golgi says that you start with the cis-golgi and with factors and proteins, this matures to the medial-golgi, which then matures again to the trans-golgi. Scientists dyed two different proteins in yeast cells – green proteins in the cis-golgi and red proteins in the trans-golgi. You can see the cell turning from green to red.
    4. Each organelle has a special function: nucleus holds DNA and does transcription, lysosomes degrade various cellular materials, peroxisomes degrade long-chain fatty acids and are important for myelin in neurons, and chloroplasts fix carbon into sugars (chloroplasts have their own genome just like mitochondria, the division of them has to be coordinated with the cell).
  1. Protein transport pathways go everywhere, where proteins start by being formed in the cytosol and then moving to the mitochondria, peroxisome, golgi complex, etc. If we look at the mitochondria, recall it has 2 membranes – the outer membrane and inner membrane, as well as the matrix. Proteins have to go to all 3 layers – proteins that enter the mitochondria have to differentiate between which of the membranes it is supposed to go to.
    1. The Smooth and Rough ER, has ribosomes that are stuck to the membrane (rough) and ribosomes that are floating around. The attached ribosomes are going to build proteins that will end up in the golgi, which are sent to membranes, and the free-floating ones will build proteins that are not membrane proteins.
    2. The ER can be studied in-vitro as microsomes, where the cell was crushed and the ER extracted, which then formed little vesicle structures called microsomes. These microsomes perform the same duties, but in little microsomes. The microsomes of interest are separated by centrifugation, and then looking for a marker protein to figure out which fraction is your organelle.
      1. By synthesizing proteins using free-floating ribosomes, they then added the microsomes. If these proteins were supposed to go in the ER, they would, but it turns out they didn't. In fact, the signal sequence on the N-terminals on the proteins wasn't cleaned – when theese proteins were by the ER, you see a clean signal sequence. Obviously it isn't as easy as proteins being channeled into the ER
        1. Another experiment was run, where the same system was used except microsomes were now present during the synthesis of the proteins – now the proteins are cleaned of the signal sequence and inside the microsomes. You have to couple translation with the transport of the proteins into the ER – both must be done at the same time.
        2. What happens is that the ribosome takes the RNA and starts to synthesize the protein. After a while, the N-terminal sequence is exposed outside the ribosome, and the Signal Recognition Particle (SRP) binds to the signal sequence on the SRP. This will stall translation as the SRP binds to a SRP receptor on the membrane of the ER. The SRP protein will then get inactivated (it has GTP) and let go, whereas a translocon will draw in the protein being made. A signal peptidase will cleave off the signal sequence as the ribosome encodes into the ER. The ribosome then leaves when it's done.
          1. The GTP cycle involves a GTPase that binds to a GDP (inactive form), and a GEF (G-Exchange Factor) will exchange a P to the GDP, making it a GTP, which activates the protein. To turn this off, you introduce a GTPase Activating Protein (GAP).
      2. If you have a protein that's supposed to be on the ER membrane, how can we keep it there? A stop-transfer anchor sequence, which folds into an alpha-helix of 20-22 nucleotides, is hydrophobic and will remain in the membrane. The positive side of the protein where the anchor sequence is on is placed on the outside of the membrane – in fact, the N-terminal is the one sticking outside. With the Type II Transmembrane protein, the N-terminal is placed inside the membrane and the rest of the protein is sticking out of the membrane. The first kind is called Type III.
      3. Hydropathy plots are used to see how hydrophobic a particular residue is (the higher score means more hydrophobic. You plot the entire residue – you then look for a 6-12 nucleotide area that's quite hydrophobic, that's the signal sequence, and usually appears at the beginning of a protein. As you follow this, you may see 20-22 amino acids who are hydrophobic, that will be the stop sequence. These things mean it's a Type I transmembrane protein due to the signal sequence and stop-transfer sequence.
        1. If we have a lack of a signal sequence, then it's not Type I. But if we have a strong 20-22 nucleotide stretch of signal-anchor sequence, we have a Type II protein. To differentiate this between a Type II and Type III, you look for a stretch of positively charged amino acids, and on what side it would show up on. Positive charge on the N-terminal is Type II, C-Terminal positive charges are Type III.
        2. A multipass transmembrane protein will have many different stretches of the stop-transfer sequences (meaning it goes through the membrane several times).
  2. A few proteins can be post-translationally moved into the ER. In this case, a translocon with the Sec63 protein complex will strart to pull a protein through the translocon. A chaperone called BiP then comes in, as an ATPase. It interacts with the Sec63 complex and becomes bound to the emerging protein coming through the translocon, and breaks its ATP to ADP, attaching to the protein. This causes uni-directional movement, and more BiPs will add on, and eventually they all come off when the protein is in.

Quick Disclaimer

The information here is mostly accurate, but should be double checked against a hard source. Please do not rely on this page for exams/midterms/finals, I will update this page with the book I read when I find it in my garage.

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