Cytoskeleton, Cell Cycle & Junctions, ECM

• • The diverse roles played by the cytoskeleton includes: cell division; cell shape; cell movement; and cell trafficking.
• • The common principle underlying the construction of all three major classes of cytoskeleton is that they are made from units & arranged into networks. This occurs by: actin, being monomeric (G-actin) or filamentous (F-actin); microtubules, being long, hollow cylinders of tubulin; and intermediate filaments, being ropelike fibres made of intermediate filament proteins.
• • Actin filaments are constructed from actin subunits by polymerisation of ATP-actin subunits. In lag phase, this process occur slowly due to nucleation for a trimer of actin monomers to form an actin nucleus. In growth phase, this process occurs faster for addition of monomers onto the nucleus, as the exposed plus-end is faster for addition & the exposed minus-end is slower for addition. In equilibrium phase, this process reaches steady-state where treadmilling occurs, as net-assembly occur at plus-end and net-disassembly occur at minus-end for polymer to maintain constant length.
• • Actin filaments are polar because actin subunits are polarised with positive and negative end, where this same orientation is maintained in filament for same polarisation.
• • Actin polymerisation is regulated by changes in free actin's conformation due to binding to polymer (with ATP-actin subunits), which forms an ATP cap. An ATP cap is formed because all actin subunits have ATP bound deep in cleft of molecule, where its hydrolysis rate to ADP is slower than the rate of actin addition to polymer.
  • Actin depolymerisation is regulated by changes in the polymer due to ATP hydrolysis to ADP. As ADP-actins have reduced binding affinity to neighbouring subunits in the filament, allowing it to dissociate from filament.
• • Actin filaments are assembled into complexes in cells by actin filament accessory proteins.
• • Actin filament accessory proteins regulate actin polymerisation & depolymerisation by: profilin, binding to G-actin monomers & concentrates them at the site of filament assembly for filament elongation; thymosin, binding subunits to change subunit conformation to prevent actin assembly into filament; cofilin, binding to ADP-actin to accelerate disassembly of filament; Gesolin, severing filament by filament conformational change & binds to plus-end; and Arp2/3 Complex, acting as minus-end on filament to help nucleate actin filament, especially as branch on pre-existing filament.
• • Arp2/3 is activated by nucleation-promoting factor at plasma membrane, which binds with Arp2 & Arp3 for new configuration on the proteins that resemble plus-end, allowing new subunits to be added onto the activated Arp2/3 (the minus-end).
• • Actin filament assembly affects the functions of complexes they assemble into by: in stress fibres, assembly is contractile bundle, allowing fibres to exert tension; in cell cortex, assembly is branched & unbranched network, allowing it to underlay plasma membrane; in lamellipodium, assembly is branched network, allowing membrane protrusions; and in filopodium, assembly is tight, parallel bundles, allowing cells to sense extracellular signals & explore environment.
• • A mode of cell migration that incorporates cytoskeleton rearrangements & cell-substrate adhesion is mesenchymal cell migration.
• • The cytoskeletal rearrangements & cell-substrate adhesion occur during cycle of cellular events underlying cell locomotion by: lamellipodium connecting to stress fibres, creating tension to brace cells against surface of migration; and focal adhesion, i.e. actin-linked cell-matrix junction, being formed behind lamellipodium, linking cytoskeleton & small transient patches to extracellular matrix.
• • The structure of actin-myosin interaction consists of thin actin filament & thick myosin filament. This interaction functions by actin sliding towards or away from each other by their bound myosin motor proteins, for contracting muscles.
• • Microtubules are constructed from tubulin monomers by polymerisation of tubulin heterodimers, with alpha-beta subunits, into protofilaments. Thirteen protofilaments are then arranged parallel to each other to form microtubules with lumen in the centre.
• • Microtubules are polar because the polarity of tubulin monomers, with plus- & minus-ends, are maintained via same orientation when they associate into protofilaments.
• • The molecular basis for dynamic instability of microtubules is the depolymerisation of GDP-tubulin end at the minus-end, occurring faster than the GTP-tubulin addition at plus-end. This is due to less affinity of GDP-tubulin for neighbouring tubulins when GTP is converted to GDP.
• • The molecular basis for dynamic instability can be regulated for microtubules to be less unstable by forming a GTP cap at the GTP-tubulin addition end, the GTP cap favours growth so will allow faster elongation. Similarly, for microtubules to be more unstable, the GTP cap can be lost by GTP hydrolysis to GDP-tubulin for rapid depolymerisation at plus-end for overall shrinkage.
• • Nucleation is responsible for lag phase in microtubule growth because it forms nucleus through pre-formed gamma-TuRC binding protein at minus-end, which allows tubulin heterodimer to be added onto the plus-end protofilament, to get out/eliminate lag phase.
• • The various roles of microtubules include: being grown from gamma-tubulin ring complex of centrosome, by its minus-end being embedded in fibrous protein matrix of centrosome; and being grown from focal MTOC in fibroblast for cytoskeleton, by MTOC anchoring & stabilising/nucleating minus-end of microtubules.
• • Different types of motor proteins mediate movement along microtubules by: kinesin, moving towards the plus-end of the microtubule while carrying cargo on its own light chain; and dynein, moving towards the minus-end of the microtubule while carrying cargo through adaptor proteins (dynactin). Both motor proteins move because ATP fuels their movement.
• • Movement of kinesin along microtubules occur by lagging leg (heavy chain 1), bound to ATP, and leading leg (heavy chain 2), bound to ADP, of kinesin binding with sites on microtubule. As lagging leg 'moves forwards' by ATP hydrolysis providing energy, it bypasses lagging leg and binds to next binding site on microtubule. This continues for kinesin to deliver its cargo.
• • Intermediate filaments are constructed by monomer polypeptide chain associating with another monomer to form coiled-coil dimer from central domain of peptide chains. Two dimers can then associate with each other in antiparallel direction, forming a staggered/antiparallel tetramer. Both dimers & tetramer become soluble subunits of intermediate filament, so filament is made from lateral association of eight tetramers per addition.
• • The construction of intermediate filaments provide mechanical strength by its addition of eight tetramers each time forming a rope-like structure, which is commonly found in cells with high mechanical stress.
• • The diverse roles of cytoskeleton in cell polarity involve cytoskeleton assisting cell movement based on cell polarity. This is because protrusions are formed at lamellipodium & pseudopodium of cells when actin polymerisation in branches is activated by Rac-GTP. This is also because of increase in myosin activity & unbranced actin bundle at cell rear when actin-myosin contraction is activated by Rho-GTP.
• • Cell polarity is important because it enables cell movement, which is essential for development of germ layers & cortical neuron layers.
• • Examples of cytoskeleton in the diverse roles associated with cell polarity include: neutrophil cell front being activated by Rac-GTP pathway when chemoattractant binds, causing protrusions in this region from short-lived activation of 2nd messengers; & neutrophil cell back being activated by Rho-GTP pathway when chemoattractant binds, causing actin-myosin contraction to assist directed movement from antagonistic Rac-activation at the front of cell.
• • The four key phases of the eukaryotic cell cycle are: G1 phase, where cells grow in size & produces organelles, cytoskeletons, some DNA & proteins for DNA synthesis; S phase, DNA synthesis occurs where chromosomes are duplicated, forming sister chromatids held together by centromeres; G2 phase, where cells grow in size & produce required proteins, for checkpoints to confirm that cells are ready for M phase; and M phase, involving mitosis (i.e. prophase, prometaphase, metaphase, anaphase & telophase), the division of nucleus, and cytokinesis, the division of cell plasma for two daughter cells.
• • Cell-cycle control system triggers the major events of cell cycle division by the increasing/decreasing cyclin levels, where different cyclin molecules are specific for each cell-cycle phase in the cell. So increase of particular cyclin concentration, through gene expressions, triggers a particular phase, while decrease of cyclin concentration, through degradation in proteasomes, signals the completion of that phase.
• • The two key mechanisms that regulate cell-cycle transition are: cyclically-activated Cdk; and ubiquitylation & degradation of Cdk by APC/C regulator.
• • Cyclically activated Cdk regulates cell cycle transition by cyclin binding to Cdk triggering Cdk conformational change. This then allows CAK (i.e. cyclin-activating kinase) to phosphorylate Cdk to activate complex completely. Activated Cdk allows cells to 'pass' the phase by activating the transcription of cyclins in latter phases, if cell conditions are met. If cell conditions are not met, upstream signals would inhibit the activity of Cdk through inhibition phosphorylation, i.e. adding an inhibitor phosphate, or through inhibitor complex, i.e. binding of Cdk to inhibitor p27 complex.
• • To proceed to next stage/phase in cell cycle: G1 cyclin-CDk checks if cell environment is favourable, if not, cells don't enter cycle; G1/S cyclin-Cdk & S cyclin-Cdk checks for any DNA damage, if minor, DNA damage is fixed but if its major damage, cell apoptosis is triggered; and G2/M cyclin-Cdk checks for DNA damage, if minor, DNA damage is fixed but if its major, cell apoptosis is triggered. It also checks for potential unreplicated DNA, if there is unreplicated DNA, it triggers apoptosis as it cannot allow DNA re-replication.
• • Ubiquitylation & degradation from APC/C regulates cell cycle transition by APC/C binding to activating subunit (Cdc20), where this complex is activated by phosphorylation. Complex then recruits ubiquitylation enzymes (i.e. E1, E2) to polyubiquitylate G1/S cyclin Cdk or M cyclin Cdk with a polyubiquitylated chain, marking it for proteasome degradation. This results in decrease in Cdk concentration, which allows transition from metaphase to anaphase during mitosis.
• • The key features of S phase include DNA synthesis for replicated chromosomes using DNA polymerse, DNA helicase & other enzymes.
• • The main events of S phase occur by: DNA helicase opening a replication fork, allowing semi-conservative replication where each original strand acts as template for new strand; DNA polymerase adding complementary nucleotide bases to template strand in new strand, by nucleotide addition reactions where DNA polymerase changes conformation to tighten around base pair when adding & to relax when moving along strand; & DNA polymerase adding bases from 5' to 3' direction, forming leading strand & lagging strand with Okazaki fragments.
• • The key features of mitosis include: prophase, where duplicated chromosomes start to condense; prometaphase, where nuclear envelope collapses & chromosomes attach to mitotic spindle; metaphase, where chromosomes are lined along metaphase plate via centromere, where kinetochores, with microtubules attached to centrosomes, attach to centromere; anaphase, where sister chromatids are pulled apart by pulling force of kinetochore microtubules; & telophase, where segregated chromosomes are pulled towards opposite centrosomes (pole), new nuclear envelopes start to form & chromatids start de-bundling.
• • Metaphase to anaphase of mitosis occurs by activation of APC/C binding to subunit (Cdc20) & being phosphorylated, which also activates ubiquitylation & degradation of securin protein, that inhibits separase. This activates separase, so it can then initiate cleavage & dissociation of cohesin proteins that hold sister chromatids together, enabling microtubule pulling forces to pull sister chromatids apart in anaphase.
• • The main event of cytokinesis occurs by cleavage from cleavage furrow forming in cell membrane in the plane of the metaphase plate, due to formation of contractile ring by myosin II & actin filaments. This ring generates force in the middle of the original cell, which pinches the middle of cytoplasm to constrict it, leading to the separation of two daughter cells.
• • The three main classes of extracellular signals that can regulate cell growth, division & survival are: mitogens, which stimulate cell division; growth factors, which stimulate cell growth; & survival factors, which stimulates cell survival.
• • Mitogen can stimulate until S phase of cell division by it binding & activating RTK, which activates Ras. Activated Ras then activates the MAPK pathway, where activated Erk activates transcription regulator A to transcribe immediate-response genes for myc. Myc then binds to regulatory regions & expresses delayed-response genes of D-cyclins, that binds & activates G1-Cdk. G1-Cdk phosphorylates/inactivates Rb, for the release/activation of E2F protein, that binds & expresses S-phase genes. This activates G1/S-Cdk & S-Cdk, resulting in positive feedback loop for Rb inhibition & E2F activation.
• • DNA damage can block cell division by signalling cell arrest in G1 or S phase. As DNA damage can be recognised by ATM/ATK proteins, which then recruits Chk1/Chk2 proteins. These then phosphorylate p53 for conformational change, which prevents Mdm2 from binding to & ubiquitylating p53 for degradation in proteasome. Accumulation of p53 allows it to be transferred into the nucleus, where its binds to regulatory regions of p21. This encodes p21 Cdk inhibitor protein that can then bind to & inhibit the activity of G1/S-Cdk, preventing cells from progressing to next phase in cell cycle.
• • Other ways for cell cycle to be halted include the overexpression of myc. This is because the excessive myc production leads to activation of Arf protein that binds to/inactivates Mdm2. This allows activated p53 to accumulate & bind to regulatory regions for cell cycle arrest or apoptosis.