| Microtubule associated proteins (MAPs) : regulators of microtubule (MT) stability and function | Plus end microtubule binding proteins (+TIPS): EB-1 Only binds GTP-tubulin - stabilises the MT seam.
DASH ring complex - Binds polymerising and depolymerising MTs (couples kinetochore movement to MT depolymerisation).
Cross-linking, stabilising or bundling proteins: MAP2, Tau - Binds side and stabilizes parallel MTs. MAP65 (Ase1) - Binds side and stabilizes anti-parallel MTs |
| EB-1 is a plus end MT binding protein that stabilises the MT seam. | EB-1 protein binds only to GTP tubulin. An EB-1:GFP fusion protein is expressed in mammalian cell which labels only the growing ends of microtubules. |
| Dam1/DASH complex | The Dam1/DASH complex is a fungal specific heterodecamer which is necessary for kinetochore-microtubule interaction in budding yeast. The purified heterodecameric complex is T-shaped and can link together to form chains. In vitro the complex can form rings around purified microtubules. The ring is composed of 16 heterodecamers with a gap of 75 A between the outside of the microtubule and the inside of the ring. |
| Dam1/DASH complex (pt.2) | The Dam1/DASH complex can bind to the + TIPs of both polymersing and depolymerising microtubules. In this experiment Rhodamine-coupled tubulin is nucleated in vitro and Alexa-488 coupled Dam1 complex is added. At time = 0, Dam1 ring complexes are seen decorating the length of the microtubule. As the microtubule depolymerises the signal at the depolymersing plus end increases as Dam1 ring complex accumulate there. This suggests it can act as a ring in vitro. |
| Dam1/DASH complex experiment | The Dam1/DASH complex can harness the force generated by microtubule depolymerisation to move beads in vitro. In microtubules are attached to a glass slide and Dam1 complex covalently couple to a bead is added. Laser based optical tweezers are used to move the bead to assess the force that can be applied on the Dam1/DASH complex without it falling off shrinking microtubule ends. |
| MAP65 | MAP65 bundles and stabilises anti-parallel MTs. This is especially important during for formation of a bi-polar spindle. MAP65 has both a MT binding domain and a dimerization domain. The configuration of MAP65 dimers allows only anti-parallel microtubules to interact. The EM image shows a cross-section through an anaphase B spindle. Serial sections through the entire spindle reveal that microtubules associate only with microtubules from the opposite pole in a stable squared packed structure. Bridges can be observed which may be MAP65 itself. |
| Long range transport of synaptic vesicles in axons requires microtubules | Several classes of proteins stabilise MTs by associating to the MT lattice by either promoting polymerisation or inhibiting MT catastrophe. The Tau class which include MAP2 and Tau itself bind via a positively charged tail which binds the negatively charged surface of the lattice. MAP2 is only found in dendrites where it forms fibrous cross-links between MTs and intermediate filaments. Tau is found in dendrites and axons and acts as a bridge between parallel MTs. |
| Motor proteins for microtubules | Kinesin: 14 structurally related classes. Includes those that can either stabilise,de-stabilise or bundle MTs. Kinesins are mostly plus end directed motors but some (such as kinesin-14) are minus end directed. Use ATP to generate force.
Dynein: Fast minus end directed motor. Structurally unrelated to kinesins. Use ATP to generate force. |
| Kinesin and Dynein motors | Kinesin and Dynein motors are responsible for numerous microtubule-dependent transport events in eukaryotic cells. The organisation of the ER and Golgi depends on the orientation of microtubules and motor activity. If colcemid is added to cells all ER and Golgi structure is disrupted. Vesicle transport is also blocked. As the microtubules re-grow the ER does so along extending microtubules, starting at the MTOC. Kinesins direct exocytosis (towards the plasmamembrane) and dynein directs endocytosis (away from the plasmamembrane). |
| Skin coloration | Regulated melanosome movements in fish pigment cells. These giant cells are responsible for changes in skin coloration and contain large pigment granules that can change their location in response to hormonal or neuronal stimulation. The second messenger in these signalling pathways is cAMP. The pigment granules aggregate or disperse depending on the concentration of cAMP in the cell |
| Mechanics of kinesin movement. | The single dimer of kinesin can move processively along the tubule because the action of the two heads are co-ordinated and one head is always bound. Thus an individual kinesin molecules can transport a cargo such as a vesicle or mitochondrion a long way - for example along an axon. |
| Steps of the mechanism | 1. Forward motor binds b-tubulin, releasing ADP. 2. Forward head binds ATP. 3. Conformational change in neck linker causes rear head to swing forward. 4. New forward head releases ADP, trailing head hydrolyses ATP and releases Pi |
| Dynein structure | Dynein is a complex motor protein consisting of two very large (500 kDa) heavy chains, two intermediate chains and two light chains. Thus like kinesin, dynein is double headed having two microtubule binding domains. The Dynein heavy chain contains an N-terminus tail which binds the Dynactin complex (to bind cargo) and a C-terminus composed of 6 AAA ATPase domains which are arranged in a wheel. The C- terminal ATPase domain closes the wheel by forming contacts with the first AAA domain which is the major ATPase that generates the conformational change which alters the position of the tail relative to the ATPase wheel. A stalk region in between AAA domains 4 and 5 binds the microtubule. |
| Dynein movement | Dynein moves exclusively towards the minus end of microtubules. However, unlike kinesin, it cannot mediate cargo transport by itself. Rather it requires an 11 subunit complex called the Dynactin complex. This contains Dynactin which also has a microtubule binding domain of its own and other proteins including Arp1 (an actin related protein) which forms a mini actin-like filament that is responsible for cargo binding. |
| d- and e- tubulin | Components of centrioles and basal bodies. Found only in some eukaryotes and in protozoa such as
Paramecium and Chlamydomonas (green algae). |
| Centriole structure | The centrosome is composed of two centrioles (a mother and a daughter) surrounded by pericentriolar material (matrix) to which g-tubulin containing MTOCs are associated. Each centriole is composed of 9 pairs of short stable triplet microtubules which contain not only a- and b- tubulin but also d and e tubulin, the functions of which are not that clear. |
| More complex microtubule structures | Singlet microtubule, built from 13 protofilaments. In some cases singlet MTs can contain 11 or 15 protofilaments. Doublet or triplet microtubules are found in specialised structures (centrioles), the latter of which form the base unit of cilia and flagella. These contain an A tubule, which has 13 protofilaments and a B tubule (in doublets) or a B and C tubule (in triplets) which have either 10 or 9 protofilaments, respectively. The additional protofilaments come from the A or B tubule. Note that the lumen of the B and C tubule is distinct. Doublets are flexible but less so than singlets and triplets are completely inflexible - particularly because they are short. |
| Actin | Actin is a globular protein (G-actin) which is divided by a central cleft that binds ATP. An actin filament appears as two strands of subunits. One repeating unit consists of 28 subunits (14 in each strand) covering a distance of 72nm in length. The filament has a clockwise helical twist such that the filament has symmetry every 36nm. The ATP binding cleft always binds to the opposite side of the adjoining actin molecule. This gives the filament polarity with the actin binding cleft exposed at the minus end. Filamentous actin is called F-actin. |
| Nucleation (lag phase), elongation (growth phase) and steady state (equil. phase) | ATP-G-actin monomers (G for globular) form stable oligomers of actin (filament nuclei) slowly. Nuclei are rapidly elongated by addition of subunits to both ends. In the third phase the ends of F-actin filaments (F for filament) are in a steady state with monomeric G-actin. This is achieved at the critical concentration Cc. Below the Cc the filament will disassemble, above the Cc the filament will get longer. If preformed filaments are added there is no lag (nucleation) phase but the Cc remains constant. |
| Rate of addition of ATP-G-actin | The rate of addition of ATP-G-actin is much (10X) faster at the plus end than the minus end, whereas the rate of dissociation is similar. In the filament ATP hydrolyses to ADP-Pi and Pi is released slowly giving rise to a filament containing ATP-actin, ADP-Pi-actin and ADP-actin. ATP-actin is added preferentially at the plus end while ADP-actin disassembles at the minus end, giving rise to treadmilling of subunits. [Note that Cc = koff/kon.] |
| Profilin competes with Thymosin for binding to actin monomers and promotes assembly | In cells G-actin is 1000x more concentrated than the critical concentration for actin filament formation. However most G-actin is bound to thymosin-b4 and this cannot be incorporated into filaments. Raising thymosin-b4 levels by micro-injection inhibits actin filament assembly by sequestering actin from dissociation filaments. Profilin binds G-actin more weakly than thymosin-b4, but stronger than actin + ends. Uniquely it allows ADP/ATP exchange and can promote ATP replacement and promotes actin incorporation into filaments. Profilin is mostly at the plasma membrane bound to PIP2 |
| Cofilin | It binds to the sides of ADP-actin in the filament, inducing them to fragment. In this manner cofilin replenishes the pool of free ADPactin which can be recharged by profilin to be used again. Actually dissociation of ADP-actin filaments occurs in two steps - the first requiring cofilin which chops off 18-20mers and a secondary step which requires Aip1 (actininteracting protein 1) which chops the 18-20mers to monomers. |
| Cap proteins and tropomodulin | The dynamics and treadmilling of actin filaments are regulated by capping proteins that specifically bind to the ends of filaments. If this didn’t happen actin filaments would grow and shrink in an uncontrolled manner. CapZ binds with very high affinity to the plus end and inhibits both subunit addition and loss. The affinity of CapZ is regulated by lipids at the plasmamembrane - so filaments are less likely to be capped near the cell cortex and thus can grow. Another protein, tropomodulin, binds the minus ends of filaments and in doing so promotes filament growth. Tropomodulin usually works in conjunction with tropomyosin (which is a rod like molecule that binds and stabilises the filament) in muscle |
| Formin | In cells the formation of actin filaments is tightly spatially regulated. Formin is a multi-domain protein containing Rho GTPase binding domain (RDB), an profilin-ATP-actin binding domain (FH1) and an filament nucleating domain (FH2). When not bound to Rho the RBD binds and inhibits the FH2 domain. When Rho GTPase becomes activated formin is recruited to the plasmamembrane. This causes a conformational shift which releases the FH1 and FH2 domains to trigger filament formation. |
| Arp2/Arp3 complex | The Arp2/Arp3 complex induces branching of actin filaments. The angle at which branch filaments are nucleated is fixed at 70 degrees . The Arp2/Arp3 complex is frequently located near the cell membrane and can be activated by proteins such as WASp. Steps: 1. the complex binds 2. conformational change follows with the activation of WASp 3. fixation at 70 degree angle |
| WASp | WASp is a multi-domain protein containing Rho GTPase binding domain (RBD), an actin binding domain and an Arp2/Arp3 complex binding domain. When not bound to Rho, the RBD prevents binding to the Arp2/Arp3 complex. When Cdc42 GTPase becomes activated, WASp is recruited to the plasmamembrane. This causes a conformational shift which releases the Arp2/Arp3 binding domain to trigger branch formation. |
| Microvilli | Microvilli are stable extensions of the plasma membrane produced by the close packing of actin filaments by villin and fimbrin which are short cross-linking proteins. If villin or fibrin are over-expressed in a fibroblast they will make microvilli! Microvilli increase the surface area of many cells, usually in polarised epithelia, like the epithelium of the intestine (up to 20 fold). This also nutrients to be absorbed more efficiently. The side arms linking the filaments to the membrane are myosin I and calmodulin |
| actin filaments with a-actinin vs fimbrin | Actin filaments can be bundled into tight or loose bundles with either the same or mixed polarity. For example a-actinin form dimers which bundle either parallel or antiparallel filaments in contractile bundles. In muscle the filaments are always arranged in a parallel fashion. The distance between the bundles allows myosin to get to the actin filament. By contrast fimbrin only bundles filaments of the same polarity and packs them more tightly. This is observed in structures such as microvilli. |
| Filamin | Actin is one of the most abundant proteins in cells and is present particularly in the cell cortex. Here it forms networks and bundles, stabilised and modulated by crosslinking proteins, such as filamin. Filamin dimers with itself in such a way that the actin binding sites are at an angle to one another. Filamin-actin networks form gels that resist rapid deformation, but adjust to tension slowly. So cells resist abrupt forces, but slowly change shape. |
