MCB L13-14
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MCB L13-14 - Leaderboard
MCB L13-14 - Details
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| 🇬🇧 | 🇬🇧 |
| Proteostasis | Proteostasis in the dynamic regulation of the protein compliment being expressed within a cell. Balance maintained via a combination of biosynthetic pathways including expression, folding and trafficking. Balance also maintained by degradation pathways too, cycling cellular material = autophagy. ER stress also a major activator of autophagic activity. |
| Autophagy | Autophagy – self-eating. Process in which cytoplasmic material is delivered to the lysosome inside a double-membraned vesicles for degradation. Conserved in eukaryotes. |
| Why do cells use autophagy? | 1. Turning over cell content under low nutrient conditions: Autophagy plays important role in cellular response to metabolic stress, contributing to maintenance of intracellular homeostasis. 2. Removal of aggregates, damaged organelle and pathogens. 3. Cell differentiation and developmental remodeling |
| Recycling and degradation in cells | Lysosome is central organelle for intracellular degradation. Hydrolytic enzymes are delivered via the secretory pathway, Activation of hydrolytic activity in the lysosome, with low pH, Contains nucleases, proteases, glycosidases, lipases, phosphatases. Delivery of extracellular and PM proteins by endocytic pathway. |
| Different types of autophagy | 1. Chaperone-mediated autophagy 2. Microautophagy 3. Macroautophagy |
| Autophagosome | Autophagosome is a unique organelle that mediates autophagy, Double membraned vesicle, engulfs targets |
| Autophagosome formation | Start with the Pre-autophagosomal structure/phagosomal assembly site (PAS). Focus of autophagy-related (Atg) proteins on membrane ER in mammals. Hierarchical action of 15 Atgs and associated complexes form the autophagosome. Complexes: mTORC1, Atg1/Unc-51-Like kinase and Class III PI3K. TORC1 as an energy sensing receptor- supresses ULK1 (also called Atg) under nutrient rich conditions. TORC1 deactivation - ULK1/Atg1complex is activated and translocates to domain of ER.ULK1/Atg1 complex phosphorylates the class III PI3K complex. This regulation is also promoted Exo84-containing exocyst complex. Atg9 vesicle interacts – possibly for membrane stability. PI3K is synthesising PI3P (PI3P recruits and activates other proteins, DFCP1 – promotes omegasome, WIPIs – maturation of omegasome and IM). Under no stress – Beclin1 in ER membrane bound, and inhibited by Bcl2. In stress – Bcl2 phosphorylated and Beclin1 joins complex. |
| Degradation by autophagy – who decides? | Receptor like proteins needed for specific cargo loading. p62 also known as Sequestosome 1 ➔ ubiquitinated targets. NDP52 and OPTN are sequestosome 1-like receptors (SLRs) ➔ bacterial pathogens ➔ xenophagy and immune response |
| Degradation by autophagy – who decides? (Bacterial recognition) | 1.Induce starvation – take nutrients. 2.Tol-like receptors can recognise bacteria and initiate autophagy. 3.Xenophagy initiated by SLRs or other mechanisms. SLRs share common motifs:Common LIR/CLIR motif – LC3-interacting region, UBx motifs, e.g. UBAN –ubiquitin binding domains. |
| Consequences of autophagy dysfunction at an organismal level | Cancer, infection and immunity, heart disease, liver disease, ageing, myopathies etc. |
| Consequences of autophagy dysfunction at a cellular/tissue level | AA insufficiency, aggregate formation, ROS production, chronic infection, apoptosis etc. |
| How to study autophagy? | Difficulty: Multiple functions of genes: Pleiotropic effects of genes. Chemetic (chemistry + genetic) approaches |
| The Cytoskeleton | Cytoskeleton refers to three main forms of protein polymer (Actin, Microtubules and Intermediate filaments) which are crucial for a multitude of cell functions including internal cell organisation, cargo transport, cell shape, integrity, cell motility and communication with the cell exterior. |
| The cytoskeleton is responsible for cellular and body mechanics | Intracellular organisation, Protein and RNA transport, Maintaining cell integrity, Chromosome segregation, Cell division, Body movement, Digestion, reproduction etc. |
| Microtubules structure | Microtubules are polymers of a-dimer containing 1 molecule of a-tubulin and 1 molecule of b-tubulin. Both the a-tubulin and b-tubulin subunits of the free a,b-tubulin dimer bind GTP (as the conc. of GTP in the cell is much higher than GDP). GTP bound a,b- tubulin dimer is incorporated into existing microtubules by binding to an exposed a,b-tubulin dimer at the end of the polymer so each protofilament has alternate a-tubulin and b-tubulin subunits. Incorporation of the dimer causes hydrolysis of GTP bound b-tubulin to GDP bound b-tubulin. a-tubulin remains bound to GTP. The a-tubulin and b-tubulin subunits also make side contacts with other a-tubulin and b-tubulin subunits in adjacent protofilaments to make sheets of ~13 parallel protofilaments which zipper together to form a microtubule. This leaves a hole down the centre of the tubule - which is refered to as the lumen. |
| A- and b- tubulin | A/b heterodimers form microtubules. Major constituents of microtubules. Found in all eukaryotes. |
| Plus and minus ends | In solution a,b-tubulin dimers can only form a polymer when above a threshold concentration, known as the critical concentration (Cc). The critical concentration for addition of a-,b-tubulin dimers at the plus end is lower than addition at the minus end so microtubules tend to grow at the plus end. Experimental evidence that tubulin is preferentially added onto the plus end is seen by adding tubulin to a bundle of microtubules from a flagellar nucleus. This shows that tubulin is mostly lost and gained from the same end (the plus end). |
| Hydrolysis and polymerisation | GTP bound b-tubulin (T form) undergoes slow hydrolysis to GDP so that GDP bound b-tubulin (D form) is the predominant form in the lattice. At the plus end since polymerisation is much faster than hydrolysis polymerising microtubules have a GTP cap. By contrast polymerisation is slower than hydrolysis at the minus end so the minus end is always in the D form. |
| Treadmilling: key concept | In many cases the minus end of microtubules is capped (see later) so that growth and shrinkage of microtubules occurs only at the plus end. However in some circumstances (such as in mitosis) the minus end is free to undergo disassembly while the plus end is polymerising giving rise to the phenomenon known as treadmilling. In vitro this occurs when Cc (T)<C<Cc (D). When the rate of polymerisation at the plus end is equal to the rate of depolymerisation at the minus end the polymer stays the same length (as on the right) but microtubule flux occurs |
| Plus ends | The plus ends of interphase microtubules are directed towards the cell cortex where they probe the inner face of the plasmamembrane (PM). Microtubule plus tips frequently pause at the plasmamembrane. This allows proteins that track the plus tips of MTs interact with proteins at the PM - including those that are responsible for nucleating actin filaments. These interactions also determine the length of the pause. In this manner MTs can direct and maintain cell shape or determine where new sites of cell growth will occur. Note that MTs can also grow along existing MTs and that single microtubules grow outwards more slowly than shrinking microtubules contract away from the PM. |
| Microtubule assembly and shrinking | Elongating filaments are straight, whereas shrinking filaments have curved or frayed ends. MT assembly is favoured when microtubule is straight. Hydrolysis of b- tubulin bound GTP to GDP causes a conformational change in the a-,b- tubulin dimer which tenses the MT lattice. The protofilaments splay outwards when depolymerising but are prevented from doing so when the microtubule is polymerising by the presence of the GFP-tubulin cap. CAP dissociation promoted explosive depolymerisation. Microtubule associated proteins (MAPs) and some motors shift the equilibrium by promoting either assembly or disassembly. |
| Dynamic instability | The cycle of microtubule polymerisation and depolymerisation is essential for the dynamic instability of microtubules observed in vivo. The rate of microtubule polymerisation, pausing, depolymerisation and rescue can be modulated by microtubule associated proteins (MAPs) and motors. Steps: 1. Rapid growth with GTP-capped end 2. Accidental loss of GTP cap 3. Rapid shrinkage 4. regain of GTP cap 5. etc |
| Drugs that cause microtubule stability. | Taxol: Blocks mitosis by stabilising microtubules, but does not block other functions of microtubules. Nocodazole: Binds to tubulin dimer and prevents its polymerisation into microtubules. Blocks mitosis by causing disassembly of the mitotic spindle and preventing chromosome segregation. Colchicine: Binds to tubulin dimer and prevents its polymerisation. Colcemid: It's structurally related to Colchicine but its action is reversible. |
| G-tubulin | Major component of g-tubulin ring complex (y-TURC) recruited to microtubule organizing centres (MTOCs) - usually the centrosome. Found in all eukaryotes. The g-tubulin ring complex (g-TURC) is composed of a ring of 13 subuntis of g-tubulin onto which a-,b- tubulin dimers bind. |
| Microtubule organising centres (MTOC) | Microtubules are nucleated throughout the cytoplasm, but are especially concentrated around the nucleus. When colcemid is added to cells injected with fluorescent tubulin, microtubules disassemble. When colcemid is removed microtubules can be seen to grow from this microtubule organising centre (MTOC). In animal cells this is the centrosome. In this manner the minus ends of microtubules are at the MTOC near the nucleus and the plus ends at the cell periphery. |
| Centrosome. | The major microtubule organising centre (MTOC) in animal cells is the centrosome. The centrosome is composed of two centrioles surrounded by pericentriolar material (centrosome matrix) to which g-tubulin ring complexes (g- TURCs) are associated. The g-TURCs are responsible for nucleation of microtubules. Recruitment of g-TURCs to the centrosome explains the nucleation pattern of microtubules seen previously. However, although microtubule nucleation always requires g-TURC, not all microtubules are nucleated from the centrosome, as seen in the epithelial cell on the right. |
| During cell mitosis | During animal cell mitosis the g-TURC complex is lost from spindle poles so the minus end of spindle microtubules can depolymerise while the plus end continues to polymerise. This behaviour, called microtubule flux, aids the establishment of chromosome bi-orientation (sister chromatids are attached to MTs from opposite spindle poles) and the speed at which they separate at anaphase. Microtubule flux can be observed by fluorescence speckle microscopy (FSM) when some, but not all, of the tubulin molecules in the cell are fluorescently labelled with GFP. During live cell imaging speckles of fluorescence can be seen to moving polewards during prometaphase, metaphase and anaphase. However, spindle microtubule length remains constant. |