Cell Biology, An Overview Pt 3

The second 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. Signaling networks are a combination of pathways – such as hormonal and neural at the same time, to stimulate a desired effect out of cells. For example, in liver cells, hormonal stimulation can activate DAG, IP3 and cAMP at the same time. This results in positive regulation of PKC, PKA, and Calcium, which causes further negative regulation of GS (Decreased glycogen synthesis), whereas GPK regulation is upregulated, leading to increased glycogen degradation.
  2. Cardiovascular diseases (insufficient bloodflow to the muscles and heart) could be treated by controlling vascular dilation, so bloodflow would be increased. Nitroglycerin was known to cause vasodilation, and thusly used to treat cardiovascular diseases. Nitroglycerin required cGMP (like cAMP). In vivo, vasodilation could be caused by acetylcholine addition. So what is the pathway?
    1. Acetylcholine added to aortas lead to vasoconstriction – but at the same time, some experiments with the aorta with acetylcholine lead to vasodilation. It turns out, with the inclusion of endothelium, you get vasodilation – and without endothelium cells, you get vasoconstriction. So it turned out the acetylcholine was acting on the endothelium each time – and that endothelium was releasing something to cause vasodilation, known as nitric oxide.
      1. The overall pathway starts with acetylcholine, leading to NO synthase which produces nitric oxide, which then travels to smooth muscle cell and causes relaxation.
    2. Phentolamine leads to muscle relaxation – which then leads to male erections. The only problem was, this drug was too broad a spectrum and could not be taken orally. And injections hurt, so to solve male erection, there needs to be an orally taken drug that specifically targets penile smooth muscle cells.
      1. The brain sends a signal down a NANC nerve cell, which cause the release of nitric oxide – only in the penis area, due to the brain only being stimulated in this region. This leads to vasodilation, which then result in an erection. One thing that needed to be done was the decreased level of phosphodiesterase – to prevent the breakdown of the nitric oxide.
      2. So this leads to Viagra – originally identified as a possible cardiovascular solution. Viagra targets PDE5, a very specific phosphodiesterase, which is only found in the penile region. Also, Viagra only shuts off a pathway to stop degradation, it does not actually initiate the cascade of hormones and whatnot for an erection. Viagra also happens to have a small effect on PDE6, found in the cone cells of the retina. This makes everything blue when you look around.
  3. A cell senses glucose without ligands – the glucose enters the glucose-sensing cell, which is converted into pyruvate, changing ADP to ATP. This will then close potassium channels (The ATP will), decreasing the membrane potential from 70 mV to 40 mV (due to lack of ions flowing, this causes membrane depolarization). A voltage sensitive calcium channel opens from this depolarization, allowing calcium to flood into the cell. This calcium makes the insulin-containing vesicle be released.
  4. Long term changes, such as to cell fates, the entire transcription profile needs to be altered. Secondary messengers like cAMP can lead to these changes – it activates Protein Kinase A, which can then travel into the nucleus, to phosphorylate a protein CREB. CREB then combines with another protein to cause transcription of CRE.
  5. Erythropoetin causes erythroid cell differentiation. This EPO on hematopoetic stem cells causes the formation of red blood cells – it uses a pathway called JAK/STAT. This pathway involves a ligand binding to a site, which activates the JAK protein. The proteins in this pathway require dimerization in order to properly activate due to monomers joining. Receptor changes conformation on the JAK proteins, which result in cross-phosphorylating one another. The active JAK then continue to phosphorylate multiple C-terminal sites on the receptor.
    1. Once this JAK is activated with the receptor, it recruits STAT proteins, which it phosphorylates. The STAT dimerizes with one another, and then it transfers into the nucleus to activate transcription and change cell profiles.
    2. JAK is turned off in the short term when it phosphorylates a phosphatase, SHP1. This phosphatase removes the phosphoresidues on the activated receptor – that means JAK is turning itself off in a way. In the long term, the usage of signal blocking and protein degradation by SOCS proteins is how EPO is regulated. SH2 binding domains on both of these proteins bind to the JAK – SOCS recruits E3 Ubiquitin Ligase after.
  1. Ub-Proteosome degradation can be used to degrade an inhibitor protein (such as I-kBalpha) so that the protein it was bound to is activated. I-kB is actually re-synthesized by transcription induced from the p65p50 compound the I-kB was bound to. This pathway is called NF-KappaB. NF-KappaB is sequestered by I-kB, and I-kB gets phosphorylated by signals, then this gets ubiquinated and degraded – resulting in NF-KappaB being free. With the Nuclear Localization Signals on the NF-KappaB, it goes into the nucleus and will result in specific transcription.
  2. The Delta-Notch pathway relies on a Notch-binding domain and a Delta-binding domain, each on a separate cell. Delta with the notch-binding domain is the ligand, and the Notch protein with the delta-binding domain is the receptor. When delta is bound to notch, you get a protease in the membrane that cleaves the extracellular and the intracellular C-terminal portion of notch.
    1. In Alzheimer's disease, there's a protein called APP in the cell membrane.- when it's cleaved, alpha secretase makes a normal cleave, but beta-secretase leaves incorrectly, leading to a Alpha-Beta22 protein in the membrane – the build up of this protein may be a possible cause of amyloid plaque in Alzheimer's.
  3. How do cells integrate the hundreds of signals that it recieves into one response?
  1. A cell needs to maintain a shape – but a shape is dynamic and able to be changed rapidly (macrophages must be able to engulf pathogens as well as move towards pathogens). A cell must also resist any malformations of the shape it's achieved. A cytoskeleton is a rigid, but also fluid skeleton for the cell. Generally, the cytoskeleton is regulated by signals from soluble factors in the membrane.
    1. The cytoskeleton is made of microfilaments, microtubules, and intermediate filaments. Microfilaments form rigid gels, networks, and linear bundles. Microtubules are fairly rigid and not easily bent, and intermediate filaments have great tensile strength.
    2. Actin microfilaments provide structures such as microvilli, cell cortex, adherens belt, filopodia, stress fibers (allow movement of vesicles along the fibers), and contractile rings. Actin is formed as G-Actin, or globular actin. This actin has an ATP binding cleft, allowing ATPase action. G-Actin can polymerize to form F-Actin, or filamentous actin, which is two interwoven chains of G-Actin.
      1. Actin makes a sort of an arrowhead – the pointed end is the (-) end and the barbed end is the (+) end. The difference is that the G-Actin will bind to the (+) end of existing actin filaments. Conceptually, we remove actin from the (-) end and add it to the (+) end. The (-) end has ADP bound actin, where a protein called Cofilin binds to and causes the breaking down of filaments at the negative end. Then Profilin changes the ADP-actin to ATP-bound actin. This would bind to the (+) end, or be bound to thymosin-B which is a sort of middleman that pauses the actin – it won't bind to existing filaments, but remains ATP-bound. Regulation of these 3 cycles lead to active regulation of the size and shape of actin filaments.
        1. If you keep removing (-) actin and adding to the (+) actin end, then your filament will move forwards.
  1. Crusher Fish have a secretory pathway that is basal and thusly very important. These fish have a mutation in the chondrocyte secretory path – chondrocytes are cells that usually make cartilage, and of course, these fish are very dependent. The mutation causes head deformities.
    1. Normally the pathway is ER to Golgi Body to Plasma Membrane, which Collagen is secreted. The mutation causes a bloated ER, abnormal golgi body, little collagen in the extracellular space, and sometimes the collagen is associated with proteosomes.
    2. Sec23A shows issues, which is important for the recruitment of coat proteins of COPII to move things from ER to Golgi. Chondrocytes, being very important early on, rely heavily on the transfer of collagen early onwards out of the cell, making this mutation very dangerous.
  2. In humans, CLSD is a condition similar to this. It also causes facial deformities and stuff.
  1. In vitro, we can visualize actin polymerization – the rate, the length of the filaments, and various other forms of analysis such as adding other material to this slide and seeing what happens. With regular G-Actin, you can study the polymerization process over time, starting with nucleation with little monomers, then elongation to a steady state. However, by starting with actin filaments who are starting to polymerize, you skip the nucleation phase and you immediately go into elongation. The nucleation step of actin is the rate-limiting step.
    1. Actin nucleators start with formins, which is a protein dimer (think two half rings) that recruits monomers of actin, and build up the actin quickly. Formins have 3 domains called RBD, FH1, and FH2. FH1 and FH2 are required to recruitment of G-actin and addition to the (+) end. Another key regulator is the Arp2/3 complex, which has the role of branching the actin out. The protein WASp comes in and binds to the activation domain of Arp2/3, and Arp2/3 will recruit actin @ a 70 degree angle to the original actin filament, creating a branched strand.
    2. In an intra-cellular pathogen, such as bacteria, may use actin as a 'tail' to propel itself around a cell. The bacteria have proteins that nucleate actin and the tail is pretty much a treadmill action of disassembly and assembly of actin monomers.
  2. For a cell to move, it has to first detach itself from a substrate in a very controlled fashion – only the rear should detach at first, and the membrane at the front should be pushed forward with structures called lamellipodium (bundles of bunched actin treadmilling) and form a new adhesion to the substrate.
  3. In the cell membrane, you have extremely important integrins that act as a bridge between the extracellular matrix and the cell itself. They can recruit a bunch of other proteins to form a focal initiation complex. And so, with the coordinated addition and removal of integrin(see slide, the cell kind of rolls along, like a ball), you get the end result of movement.
    1. The leading edge of the cell has to have branched actin treadmilling occuring. There's also stress fibers, which have contractile bundles that contract and help to move the cell by squeezing it like a tube of toothpaste.
  4. Cells are directed by proteins that help to polarize a cell to drive the direction of cell movement. In general, receptors for a cell are not polarized, because you want a cell to sense signals from all directions – then you have proteins localized to the leading edge or the back end of the cell (back end proteins like PTEN may help remove adhesions and squeeze the cell towards the leading edge).
    1. A few GTPases are involved in regulation of the cytoskeleton – such as Rho, Rac, and Cdc42. Each one is involved in a different formation of a structure, where Rho mutants that are superactive form stress fibers, whereas Rac will show mostly formation of actin at the edges.
      1. Rho is important in the regulation of Formin, involved in the nucleation of actin. Rho itself is a small GTPase anchored to the membrane, similar to Ras. When Rho has a GTP bound to it (Usually thanks to a GEF), it will recruit Formin, which changes conformation and starts the recruit Profilin-ATP-Actin.
      2. Cdc42 acts on WASp, and Cdc42 functions the same way as Rho, more or less. GTPase, GEF, GTP activates, etc. Usually not just one protein drives the formation of structures, meaning the combination of all of these protein factors give the results that should be occuring. The coordinated regulation of these protein ultimately give you the cytoskeleton phenotype you look for.
  1. What mediates the shaping of actin filaments? The actin cross-linking proteins – for example, fimbrin, will form microvilli and focal adhesions. Alpha-actinins form dimmers to cross-link actin, important in stress fibers and filopodia. Spectrin forms a giant branched network that looks rather radial. Filamins cause leading edge stress fibers – from this you can see multiple proteins can be mixed together. And lastly, dystrophin link membrane proteins to actin in muscle.
    1. The motor proteins, known as myosins, come in 3 flavors. Class I, which has a single head, are important since they attach to the actin cytoskeleton and the membrane. Class II motors have two heads, which shift crossbundles of actin and can contract them towards one another. Class V myosins are tethered to vesicles and move along the actin track, bringing an organelle/vesicle with it. Myosins always move to the + end.
    2. A myosin has a neck region, and a head region. The neck region acts a lever, where the longer the lever, the more easily it is to do work. The tail region of the myosin can bundle multiple myosins together, or tether an organelle to.
    3. A lot of myosin mutations give rise to deafness and shit. Very related to disease.
  2. Myosin binds and hydrolyzes ATP to move. It binds ATP, releasing the head from the actin. Hydrolysis ‘cocks’ the head, and then the head binds to the actin filament once more, and when the free phosphate is released, the myosin strokes and moves the actin filament towards the – direction, while the myosin moves to the + end.
  3. Myosin motors can move organelles around in the cell – you can select which organelles/cargoes the myosin will move by phosphorylation or likewise scenarios. This is especially important during cell division to ensure equal distribution of organelles. The myosin used here, Class V, has two heads. Only one head becomes unattached at any one time, and it kind of ‘walks’ along the actin filament. In the muscle cell, the myosin heads and bundled together between actin filaments – the myosin ‘contracts’ by pulling towards the middle where the + ends are.
  4. Actin microfilaments can be stabilized with proteins – using Cap proteins, like CapZ, can bind to the + end of the actin microfilament and stabilize it. On the other hand, tropomodulin can bind to the – end of the actin microfilament so it won’t fall apart. A muscle has caps on both ends to ensure the whole structure stays together and does not fall apart.
  5. Microtubulins are made of a dimers – alpha tubulin and beta tubulin. A-tubulin is always GTP-Bound, but B-tubulin can cycle between GDP and GTP forms. This allows for the formation of microtubules – they line up alternating, to create a protofilament. One single circlet of protofilaments make a singlet, then you also have doublets and triplets.
    1. Microtubules can also treadmill, where addition happens at the + end and removal is from the – end. Microtubules, as opposed to actin, will not self polymerize – they need a MTOC Nucleation Center, which form a nucleus for microtubules formed from, such as a centrosome. Cell division requires two of these centers. Another example is in the axon, where microtubules are in axons – there’s also free-floating sections of microtubules in the axon as well.
    2. Basal Bodies are important for forming cilia, which are used to move the cell or move material in the extracellular media.
  6. The centrosome itself is composed of two centrioles perpendicular. y-Turcs allow for the formation of a ring structure for the alpha and beta subunits to assemble from. Y-Turcs are the beginning of a – end.
  7. Microtubules are extremely dynamic and are constantly growing and shrinking – rescue for growing, and catastrophe for shrinking (these terms only apply to the + end). The ends are curled during catastrophe, and blunt during rescue. GDP-bound B-Tubulin facilitates catastrophe, whereas GTP-bound will facilitate rescue. As a microtubule grows, only the very end of the tubule still has GTP-bound B-Tubulin, keeping it together; if you stop the addition of the GTP-bound B-Tubulins, then the subunits will begin to curve and they will fall apart. B-Tubulin will only hydrolyze GTP when it is associated with A-Tubulin.
  1. Kinesin-13 and Stathmin bind to the + ends of microtubules and drive depolymerization at the + end. Microtubules are bundled in specific ways. Proteins called Microtubule Associated Proteins are associated with microtubules – they help the spacing of the microtubules from one another. MAP2 may provide long spacing, and the Tau protein will be shorter. Moreso, some proteins called TIPs are only present on the + end of growing microtubules. These microtubule dynamics are important to understand due to how we can form different structures.
    1. There's even specific motors for microtubules. Kinesin-1 is the conventional protein, along with Kinesin-2 which both move organelles to the + end of the microtubule. The Kinesin-5 has two myosin-like heads to slide microtubules of both sides. And Kinesin-13s are the ones that break apart microtubules at their plus end. Kinesins work like myosins, using ATP force to move stuff, however, these dissociate when they're ADP bound, and when ATP binds, the 'unbound head' swings forward, using the energy from ATP → ADP + Pi. X-ray crystallography can be used to distinguish 3d models of proteins.
    2. There's another motor protein that can move towards the – end, called Dyneins. Dyeins have complexes that form and allow them to bind to specific cargo and move to the – end. So when you put this altogether, you have the organizing center, and you need to move stuff in all directions, and using your specific motor proteins, can couple to and move them. Note that with sometimes with these motor proteins, dyneins and kinesin can bind to the same organelle and 'tug of war' on some organelles. Motor proteins can also be phosphorylated., changing their activity and the direction of which organelles move.
  2. You can specifically knock out proteins that are on say, the kinesin binding to the organelle, thus downregulating the kinesin.
    1. Coordination between actin and microtubules together is what drives cell organelle movement. Both will show directional polymerization: the actin has proteins that give polarity, same with the microtubules.
  3. Cillia are micro-tubule based – made up of microtubule filaments and associated proteins allowing polymerization (think centrosomes). Basically you have a microtubule organizing center that causes polymerization into a cillia. Cillia beat, which gives external movement to the entire cell.
    1. Cillia allow movement of molecules in the cell surroundings, as well as the ability to move around mucous that is near cells. Cillia also moves morphogens – proteins that are essential in development to transition from one cell fate to another. All the cillia in a developing embryo beat in the same direction to move all the morphogens in one direction.
    2. How do cillia bend? Proteins called nexins prohibit movement in one direction – causing the molecules to bunch up in one direction, and the bending of the cillia occurs. Without the nexins we don't see any beating, instead the microtubule strands in the cillia just slide around aimlessly.
  4. The third kind of structural, intermediate filaments, depending on the particular cell type, the base protein that the filament is composed of is different. Keratins, primarily present in epithelial cells, are called Class I. Class III would be GFAP, Desmin, Vimentin which all give integrity to muscle cells and mesenchymal cells. Neurofilaments are essential for the orgnization of neurons. Lamins make up the nuclear lamina and are found in all nuclei – giving the nucleus structure and organization.
    1. These filaments have an N-terminus, and a C-terminus. These polymerize together and stuff into complex structures, and can also organize into different bundles to give rise to different structures. You can even crosslink microtubules and intermediate filaments together.
    2. Mutations in the intermediate filaments may lead to fucked up epidermis layers, since the keratin filaments are messed, leading to structural collapse and gross skin.
  1. Adhesin proteins target and locate the host cell, or the bacterium can use Type III Secretion System, where it injects a receptor into the host cell via a needle mechanism, which will then facilitate binding on the bacterium.
  2. The bacterium is then moved into the host by the zipper mechanism, which has the bacterium engulfed, or the trigger mechanism, which bacterium that use the Type III are engulfed by. Both involve the bacterium being engulfed.
  3. Once inside the cell, the bacterium can use the cellular components and processes to its own advantage. Cell adhesion molecules like clathirin can be affected, or even cellular pathways like the Rab pathway can be stopped.
  4. Lastly, when the bacterium has replicated and is ready to leave, it must avoid the autophagy process of the cell. The bacterium Listeria produces ActA, which disguises it from ubiquination.

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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