Cell Biology, An Overview, Pt 2

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. Golgi body is derived from ER – how can the cell differentiate what stays in the cell, and what goes to the golgi body? This means another layer of sorting must occur. The hypothesis states that by adding another signal sequence we can 'tag' the protein – we'll call this the Golgi sequence. There is therefore a receptor that recognizes the sequence and sends the protein to the Golgi.
    1. We call these targeting sequences. These sequence elements are sufficient to drive proteins to their correct sites. Receptors/Recognition proteins can then read these sequences and sort the proteins accordingly. If one were to take a cytoplasmic protein and then add a KDEL sequence along with a signal sequence, you can move it from the cytoplasm into the ER. Another important signal sequence is Manose 6-Phosphate – it takes the proteins tagged with it and sending them to the lysosome (usually lysosome enzymes). Speicific coating proteins coat the vesicles that hold these proteins for transport, which help target their location.
  1. The location of where secretory proteins are made in a cell can be determined by adding detergent to dissolve the ER membrane in microsomes, the proteins could be digested by proteases. However, without detergent, we don't see any digestion of the secretory protein. This means the proteins are not existent outside of the ER microsomes.
    1. A scientist from UCSD named Palade wanted to study how proteins moved through a cell and secreted, but lacked the technology to do so. Back in his day, we can use EM and high resolution microscopy. Today however, we can do epitope tagging and immuno-EM.
    2. What he did was use 'Pulse-Chase' - he gave cells a 'pulse', or a radiolabeled amino acid (in this case, he used Leucine). Proteins that are going to be made will have radioactive Leucine. Then, by washing away the leucine, only proteins made in a small window of time will be radiolabeled. He then 'chased' the cells, or stopped them to look at them with EM * Radiography and several points in time. Now he can see the progession of the newly synthesized proteins in the cell. He could see that the proteins went from the ER, to the golgi, to vesicles moving out of the golgi, then heading out to the plasma membrane. He picked a tissue that secreted the proteins – why? If the proteins went everywhere in the cell, you would not be able to tell which proteins were going where.
  2. Signal sequences allow to look if a protein is localized somewhere – assuming you have an epitope tag on it. If you happen to say, knock out a protein that is important for transport into the ER, then you won't see any movement into the ER. Let's say a mutation was made in the translocon that would not allow transport to occur, leaving proteins immobile. Using a SiRNA screen, you can mutate genes to see if one of these genes you mutated affects the transport of proteins (localization).
  3. There is no traffic between the ER-Golgi pathway and other organelles. So how does the cell send proteins to organelles like the mitochondria and nucleus?
    1. In the nucleus, the nuclear envelope has nuclear pores. These allow the passage of small ions and glucose, etc. However, how do the large proteins get into the nucleus? We need RNA polymerase, which is manufactured in the cytoplasm, in the nucleus. Specific proteins and factors can interact with the nuclear pore to open and expand it.
      1. A NLS, or Nuclear Localization Signal, is used to drive a protein into the nucleus. Another GTPase cycle is how nuclear transport actually takes place. In the cytoplasm, we have 'nuclear import receptors' which recognize proteins with NLS, which they bind to. The cytoplasmic filaments then shovel the protein into the nucleus, where a protein called Ran (A GTPase bound to GTP in the Nucleus. If it's bound to a GDP, it's inactive) will separate the nuclear import receptor from the protein and bind to the import factor itself. The nuclear import factor then gets out of the nucleus again, and now that both the receptor and the Ran-GDP are in the cytoplasm, a Ran-GAP in the cytoplasm will break down Ran-GTP to Ran-GDP, inactivating it.
        1. No one knows how the Ran-GDP can go back into the nucleus. Ran-GTP can't get out on it's own.
    2. Now if we have a protein with a Nuclear export signal in the nucleus, it will bind to the nuclear export receptor with a Ran-GTP, and then it will move out into the cytoplasm. Ran-GTP will hydrolyze into Ran-GDP and dissociate from the Nuclear Export Receptor, which then cannot bind to the protein and will release it. Ran-GDP then makes its way back into the nucleus, while the Nuclear Export Signal interacts with cytoplasmic filaments for import back into the nucleus.
  1. Endosymbiotic theory discusses how mitochondria used to exist outside of cells. Mitochondria can fission and fusion, have their own DNA, their own ribosomes, and their own proteins. All known mitochondia protein encoded by the mitochondria are not exported. It seen that mitochondrial DNA does not recombine. Usually, from the fusion of gametes, the DNA crosses over and recombines, etc, blurring the ancestry. Another thing to note is that mitochondria are maternally inherited – in a random manner via the cytoplasm (when the gamete splits, whatever mitochondria are in that part are what you get). Paternal mitochondria are degraded.
  2. Because of all this data, we can use mitochondrial DNA as a record to build phylogenetic trees. There's a view that humans all came out of Africa – the oldest mitochondrial DNA records come from Africa.
  3. When you have a protein made by a ribosome, you have to first note the ER signal sequence. ER translocation is cotranslation, meaning before the protein is even completed, if it has the signal sequence at the proper position, it will get translocated to the ER. The ultimate goal of such a protein is to be secreted out of the plasma membrane.
  4. The transport from the ER to Golgi is the COPII vesicles, and the Golgi can attach KDEL signal sequences to proteins to send them back to the ER via COPI vesicles. If there's M6P, then the protein is sent to lysosomes. ER Signal Sequence has priority over the NLS (To the Nucleus, Import Sequence).
  5. If you have a protein with a NLS (Nuclear Localization Gradient), it will diffuse into the nucleus attached to Ran-GDP, which will disassemble and Ran-GDP will become Ran-GTP, and when it moves out, it will disassemble into Ran-GDP again. This is actually correct in comparison to the lecture slide.
  1. In the cytoplasm, if you have high calcium levels, you can get dephosphorylation of a protein that exposes its nuclear import signal. This leads to the protein moving into the nucleus, where it activates gene transcription of its target genes. Once in the nucleus, the lower level of calcium will cause phosphorylation as the calcineurin phosphatase unbinds, which then exposes the nuclear export signal, and it goes back into the cytoplasm and transcription is turned off.
  2. One of the most critical things to move out of the nucleus is the mRNA. The mRNA comes out thanks to specific proteins that bind mRNA when it's in the nucleus. Once the mRNA moves out of the nucleus, the new environment causes the proteins to unbind and they go back into the nucleus.
  3. By using an uptake targeting sequence on mitochondrial proteins, you can add mitochondria to this mixture. The proteins are taken up by the mitochondria, then the addition of trypsin, which is a protease, would not degrade the proteins. If the mitochondria don't take them up, then they'll get degraded obviously.
    1. Proteins enter the Mitochondria in a very similar way to the ER. The mitochondria can end up in the outer membrane, inner membrane, matrix, or inner membrane space. A protein of this sort must not only go in the mitochondria, but target one of these locations. For instance, if a protein wants to go to the matrix, it needs a matrix-targeting sequence. If the mitochondria sees this, then it'll move the protein into the matrix. Of course, this protein must go through the outer and inner membrane – using an import receptor on the outer membrane recognizes this sequence and opens a translocon for the protein. The inner mitochondrial membrane will also open and allow passage. In the matrix, a protease will cleave off the signal sequence and there's your final protein in the matrix.
      1. The intermembrane space is special – it can use a matrix targeting sequence AND an intermembrane-space-targeting sequence at the same time. Alternatively, proteins can have a general targeting sequence for the outer membrane. With the first set of targeting sequences, the outer membrane moves the protein in. Then the inner membrane will begin to let it through – until it notices the intermembrane-targeting sequence. A protease will cleave off the intermembrane-targeting sequence, and the protein will be released into the intermembrane space.
      2. The inner mitochondrial membrane has 3 pathways – the differences are just between the targeting sequences. A matrix targeting sequence and a hydrophobic stop-transfer sequence is one way. Another is having the matrix-targeting-sequence and internal sequences to be recognized by a protein called Oxa1. The third method uses internal sequences that will be recognized by Tom70 receptors and Tim22 complexes.
        1. The first is the simplest – the outer membrane allows the protein in, and so does the inner membrane, until the hydrophobic sequence is seen. Then transfer stops, and a peptidase will cleave off the matrix-targeting sequence and you have a protein now stuck in the inner membrane.
        2. The next protein makes it to the matrix, until Oxa1 finds the protein in the matrix and inserts it back into the inner membrane.
        3. The last method has Tom70 receptors recognizing the protein from the outer membrane, sending it to Tim22 which will incorporate it into the inner membrane.
    2. In chloroplasts, you have the thylakoid's inner space called a stroma. The proteins that move in here have a thylakoid targeting sequence and a stroma-import sequence.
  4. It would be important to remember that signal sequences and signal sequence receptors do a great deal in sorting proteins. The ER-Golgi secretion pathway is very dynamic, and different signal sequences will take proteins to different locations. There are many methods to read starting sequences and to move proteins where they are supposed to be – like the GTPase cycles.
  5. Membrane itself is synthesized at a location different than the membrane itself, such as within the ER and into the Golgi before the membrane. The membrane must also be degraded, and proteins in it removed as well.
  • At close ranges, fluorescent microscopy can no longer be used. Trying to discern a protein from organelles that are 50 nm apart means you need to use immuno-TEM, electron microscopy, and super resolution microscopy, as well as cell fractionation followed by markers. Super resolution microscopy can be used on live cells to see localilzation of proteins.
  • Protein H is a Type II membrane protein – Ab2 which binds to the c-terminus, cannot bind to this protien. On the other hand, Ab1 can bind to the n-terminus. Ab2 must be located in the cell, and Ab1 must be sticking outside since the cell now has a black color. The gray colored membrane means that Ab1 and Ab2 was able to bind since Ab2 could move into the cell.
    • The relative orientation of proteins from transport to transport is the same – if a protein has he Nterminus located within the organelle, then it'll keep the Nterminus within the organelle after forming a vesicle and moving the protein around. C-terminus is always located in the cytoplasm for a Type I Transmembrane Protein, as well as a Type III. Type II has the c- terminus located in the ER lumen.
  1. How to analyze experiments using techniques discussed in lecture.
  2. Limitations of techniques, as well as alternative approaches.
  3. Concepts, but not details. Important differences of O and N glycosylation, but not anything about details.
  4. Types and functions of ATP pumps, types and functions of channels, and types and ID of different classes of TM proteins.
  5. Think of general principles and how these principles can be modified for specific needs – such as putting a protein in a membrane to targeting proteins into a Mt vs ER membrane.
  6. Synthesis of concepts into wholistic understanding is also important, such as the Ub-proteosome system applied to Hmg-CoA reductase.
  1. When you have large proteins/complexes that cannot enter the cell on their own, such as using channels or transporter proteins. You also have to move plasma membrane proteins out of the plasma membrane, such as receptors that must be downregulated. Proteins can be secreted in a regulatory fashion or constitutively (which means it’ll keep secreting regardless of environment). Things to be degraded get trafficked to the late endosome and then sent to the lysosome. Some proteins are active only on a plasma membrane – moving them off would deactivate them.
  2. A classic way to study endocytosis (used to traffick large molecules into the cell – such as cholesterol, which is never found as cholesterol, but LDL particles instead. In order for a cell to take up cholesterol, it must ingest a huge-ass LDL particle instead) was using pulse-chase, where using LDL-ferritin particles were the Pulse, following with the chase. This showed that these LDL-ferritins moved in from the membrane into lysosomes in the cell when it was time to be degraded, thanks to electron microscopy.
    1. There's a balance between specific uptake of materials, and non-specific uptake. Cholesterol is one of these materials that needs specific endocytosis. Once something gets into the cell via endocytosis, it must be sorted. Sometimes the receptor must be recycled to the plasma membrane – a sorting step will separate ligand from receptor, and the receptor can be sent back.
  3. We have adapter proteins, like AP2, which are recruited to other proteins on the plasma membrane – for instance, the LDL receptor WITH a LDL bound to it (and only with the LDL bound to it) will get AP2 to bind to it and start to form the vesicle with an accessory protein that also binds. The receptor will sit on the plasma membrane and not get encytosed if there’s no LDL. This is specific uptake – without the LDL receptors on its surface, LDL won’t be uptaken.
  4. Cargo binding can be regulated – ligand binding can be a source of influence for the receptor, which is what happens with LDL. Phosphorylation status of a protein on the membrane can also be a regulator, as well as ubiquitination. Ubiquitination can be mono, meaning just one ubiquinone binds to the protein in question, changing conformation, instead of binding a whole chain and causing degradation.
    1. For endosomes, Clathrin is the coat protein, recruited by AP2. Also, similar to adaptor proteins, accessory proteins will interact with AP2 (help recruit AP2). Clathrin is analogous to COPII and COPI we've seen previously. Of course, like the coat proteins, the Clathrin coated vesicles must also be uncoated later on.
      1. Clathrin forms a triskelion structure (a polygonal basketlike structure) - it only requires an increase in local concentration to form coating – adapter proteins help bring the triskelions together, which they fold over the vesicle. Clathrin binding to the membrane causes an invagination of the membrane, but it cannot pinch off the vesicle. HSC70 and Auxilin will cause the disassociation of the Clathirin molecules from the vesicle and leave it unbound.
      2. Dynamin is a protein that will self-assemble into a helical protein structure, which helps it pinch off vesicles. With GTP, the helix shortens its diameter. Very often these proteins will get used especially in the function is similar. The dynamin is attracted by the curvature of the membrane, weirdly enough. In-vitro, using dynamin, you can get it to bind to vesicles as wrapping around tubules, but it will not constrict to bud the tubule off into vesicles because of the lack of GTP.
        1. Dynamin is also important in the division of mitochondria. It forms a helix around the mitochondria, and once if hydrolyzes GTP, it breaks the mitochondria into two.
  5. If we follow the pathway we've been mostly looking at so far, the early endosome after the coating is taken off, binds to the late endosome with an acidic pH, leading to the LDL unbinding from the LDL receptor. The LDL receptor buds off again and moves to the plasma membrane again, whereas the LDL is moved to the lysosome degrades it to fatty acids, cholesterol, and amino acids.
  1. Looking at LDL endocytosis, AP2 recruits Clathrin, which then uncoats, and the uncoated vesicle can fuse with the late endosome, with a low 5.0 pH. This causes the LDL Receptor, which is pH-based, to unbind, and this is sorted into the plasma membrane. The LDL itself is degraded and the contents used by the cell.
  2. Free iron in the cell is toxic. A protein called Ferrotransferrin binds to iron, which is then released into the cell. In the late endosome, the iron dissociates due to the pH, and the receptor-ferrotransferrin is sent back to the plasma membrane, in which the ferrotransferrin will then dissociate at neutral pH.
  3. The M6P Pathway – M6P is a glycosolation modification. With the M6P receptor, it will bind any molecules with M6P modification. M6P is unique in that, the receptor is separated from the M6P-tagged, but then the M6P Receptor has two fates. It can merge back into the golgi instead of going to the plasma membrane – because there might be cargo in the golgi with M6P tagging that need to go the lysosome. This means the M6P receptor can move cargo from the extracellular space, or from the golgi directly to the lysosome.
  4. Via the endosomal pathway, the late endosome can do a bit of sorting – by forming a multi-vesticular body for sorting. The MVB sorts proteins into separate compartments. It uses a large vesicle to carry little vesicles – all the proteins on the membrane of the late endosome get sorted into two membrane compartments – either on the outer surface or inside vesicles in it. Everything on that membrane will get incorporated into lysosome. By forming little vesicles within the MVB, we can separate populations of proteins. Proteins that shouldn’t be degraded are kept on the membrane of the late endosome.
    1. The actual sorting process – HRS proteins on the late endosome recruit the ESCRT complex, and HRS will only work when monoubiquinated. ESCRT will cause the membrane to invaginate and pinch off to form the MVB – it also puts all of the mono ubiquinated proteins into the invagination – that’s the tagging for degradation. All of the ESCRT proteins then dissociate, and everything ubiquinated is going to be degraded. There’s also the possibility of trafficking out of the MVB into the golgi/PM if they’re to be recycled.
    2. ESCRT complexes can be abused by viruses. Viruses have monoubiquinated proteins similar to HRS that will recruit ESCRT complexes to cause budding. Then the virus goes to infect other cells as it pleases.
  5. It’s important in certain cells, like the stomach, that proteins are sorted to the right side – apical or basolateral depending on the protein. Cl- channels must be on the basolateral side so the apical side (stomach) can be acidifed, whereas Glucose absorption requires glucose symporters on the apical side. We suspect that the golgi/trans-golgi kind of helps sort where these proteins should go to maintain the polarity of the cell.
  6. Proteins must also leave cells – like hormones and neurotransmitters. Exocytosis comes in two flavors – constitutive, which is constant, or it can be regulated. Neurotransmitter release is very important, herpy derp derp.
    1. The neurotransmitter process in neurons requires a vesicle to be shuffled into the cell, filled with the neurotransmitters (sorting of cargo, specificity), targeting the vesicle to specific locations to be released properly, and then released via SNARE.
  1. The Glut-4 Receptor is activated thanks to insulin – Glut-4 is supposed to get cells to take in glucose and convert it into glycogen for storage. Glut-4 is kept in vesicles within the cell, not allowing glucose to enter the cell, along with RAB proteins that are stimulated by glucose indirectly (thanks to Insulin) – the presence of glucose causes proteins to phosphorylate and remove the RAB proteins, allowing the Glut-4 to move to the membrane.
    1. Membranes are identified from one another (endosomes, lysosomes, plasma membrane…) by the composition of RAB proteins in the membrane. Specific RABs are in specific membranes. There are 70 different RAB proteins in different patterns, leading to identity. RABs themselves are all GTPases. For instance, if you found only RAB-7 on a compartment, that’s a lysosome. With 7 and 9, that becomes the late endosome.
    2. The RAB must be removed and added to induce different membranes. If RAB is free-floating in the cytoplasm, it's bound to GDP to keep it inactive. It binds to a REP, which carries it around. RabGGT adds a lipid tail to the RAB C-Terminus, and then the REP adds it to the membrane it should be on – the lipid tail inserts it into the membrane. The GDP must then be removed, thanks to a GEF that switches the GDP for a GTP. The GTP-active RAB will recruit effectors – until a GAP breaks down the GTP to GDP to inactivate it. GDI can remove the RAB from the membrane.
    3. RABs recruit a variety of effectors: such as for cargo selection/coat formation, transport (this recruits motor proteins to drive the vesicle along a ‘track’, like a filament), tethering (this allows the membrane to stick to a target membrane) and fusion (proteins that help mediate the fusion of membranes together).
      1. The RAB Cascade hypothesizes that a vesicle tagged with a certain color protein swapped for another RAB tagged with another color – changing the identity of the compartment as well. RAB A recruits RAB B (it also recruits the GEF for RAB A of course), who recruits RAB C. This cascade changes the identity of a membrane over time – a simple model. At the same time – the RAB B will recruit the GTPase for RAB A – it will hydrolyze the GTP of RAB A and cause it to disassociate. RAB proteins are important in ER → Golgi → Secretion pathway.
      2. Specific SNARE proteins are the actual machinery that allow membranes to fuse – they only work in a complex. One part called the V-Snare is always present in a specific vesicle, and a T-Snare is present in the target membrane of the membrane. The vesicle docks to the membrane, and then the SNAREs fuse them together. The membranes are brought together in the first place thanks to RAB – which those RABs were brought to it by RABs. They help the target dock with the receptor, and the SNAREs will force them to fuse with each other.
        1. The V-Snare T-Snare complex is separated by the SNAP complex, and with ATP Hydrolysis, pop goes the complex.
  2. Large protein aggregates form from damaged proteins in a cell – this of course is no good. Also fuck damage organelles. However, these things are really big, and can't get into the ER for targeting because the proteosome won't even fit it. Autophagy is how cells get rid of this shit.
    1. Assuming a damaged organelle or whatever, the cell creates a membrane around the protein or cytoplasmic component in question. The autophagic vesicle forms around the component – the autophagic vesicle is targeted for fusion with the lysosome. This process is incredibly important.
  1. We differentiate cell signaling into short term due to stress in the environment, and long term that may affect the whole lifespan of the cell. In cell signaling, you have the release of the signal, followed by the responding cell that changes its metabolism, moves, etc. It can even lead to modification of gene expression and development. Remember, if you turn something on, you must also turn it off.
    1. When a cell responds to specific molecules, we have 4 categories. Endocrine signaling has the signaling cell secreting molecules that are carried to a distant location via the bloodstream to its target cell. An example would be insulin. Paracrine signaling has a secretory cell that is very close to the target cell. Autocrine signaling has the signaling cell being the target cell at the same time. Cell-cell signaling uses ligands on the plasma membrane of the signaling cell reacting with receptors on the plasma membrane of the target cell – they're in direct contact.
    2. You have a signal (ligand, hormone, nutrient, etc), which then goes to a receptor and binds to it. This external signal must then be transduced into the cell – via second messengers that are a sort of middleman inside the cell. This then leads to the effectors, which give the final effects of the signal.
    3. One signal can affect multiple cell types – with the same second messenger, but different effectors, then the signal can have different effects on these cells.
  2. A classic signaling pathway would start with G-proteins, then lead to a G-protein coupled receptor/sensor, and this leads to many diverse outputs. The general cascade begins with the activation of receptor, leading to a conformational change. This binds to a G-alpha subunit nearby in the cell – which causes the g-alpha protein to dissociate into a G-beta and G-Alpha, the G-alpha being the part of interest as it loses GDP and picks up a GTP, and binds to the effector, activating it. Hydrolysis of GTP on the G-alpha deactivates it to rebind to G-beta.
  3. The G-beta will also change a cell state, such as leading to membrane polarization/ion transfer binding. So while the G-alpha can bind to an effector, so can G-beta.
    1. On a G-protein called G-alphaS, the effector is adenylyl cyclase that leads to CAMP increase, whereas G-alphaI will lead to a CAMP decrease using the same effector. Although the professor doesn't want us to memorize all of these, know the concept that there are differences in the g-proteins and they will all have their unique bindings and shit.
    2. The technique FRET allows us to study protein-protein interactions. If you want to study protein interaction, you epiptope-tag one of these proteins with a fluorophore, and you tag another with a different fluorophore (we'll use CFP and YFP). If we bind these to say, G-Beta and G-Alpha… because the fluorescence energy will be transferred from CFP to YFP, we can use the excitation light wavelength of CFP, where the fluorescence energy of CFP hits YFP, leading to YFP displaying light instead. If these two proteins are not incredibly close, you see no effect. So if the G-proteins are together, we get yellow light, and when the proteins are active (dissociated) we get cyan light.
  4. CAMP (the product of G-AlphaS, and inhibited by G-AlphaI) activates Protein Kinase A – PKA will phosphorylate various other proteins in the cell. Reguatory subunits are bound to the catalytic subunits – however, cAMP binding causes release of the regulatory subunits, leading to activation. In adipose tissue, you see hydrolysis of triglycerides. In kidneys, you see absorption of water.
    1. In order to turn off cAMP signaling, you have the ability to turn it off in the short term, and the long term. In the short term, you have an enzyme called phosphodiesterase that breaks up cAMP. One of the targets of PKA, released by the cAMP, is PDE (phosphodiesterase) that breaks down cAMP. Once there isn't enough cAMP floating around, the PKA is bound to the regulatory subunits again.
  5. So why do we have signaling cascades? You get a strong amplification effect – with just one signaling molecule, we can maybe activate 3 secondary messengers, which produce 10 each of the secondary messenger molecule, which then activates so many protein kinases, which activate mutliple enzymes. We have less signaling molecules because we would need too many receptors on the cell surface to bind it, and so it wouldn't activate other cells randomly, as well as keeping osmolarity in balance. Most importantly, smaller amounts of signaling molecule allow a gradient where larger amounts can have different effects than smaller amounts.
  6. Another example involving G-Proteins involves calcium as a second messenger. The G-alpha goes for phospholipase C that breaks up a molecule called PIP2 into IP3 and DAG. IP3 will activate a IP-gated calcium channel in the endoplasmic reticulum – so calcium is released into the cytoplasm, activating Protein Kinase C. PKC will then go phosphorylate a bunch of substrates – and also open store-operated calcium channel. Calcium is the third messenger, and IP3 is the second messenger. Same protein family, completely different second messenger, a third messenger, and completely different receptors.

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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