Kinesin motors in the transport along microtubules. Dynein Motor

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1 Molecular Cell Biology Autumn 2014, Michael Pavlov Based on Chapter 17 and 18.(1-4), MCB book Lecture 9: Cytoskeleton Microtubules: Tubulin Structure of tubulin dimer and organization of tubulin dimers in microtubules. MTOC. MT dynamics in vitro. Dynamic instability of MTs Microtubule associated proteins (MAPs). Kinesin motors in the transport along microtubules Dynein Motor Roles of Microtubules and Kinesins in Mitosis. The structure and dynamics of centrosomes in the cell cycle. Complex structure of Kinetochore. MT-Kinetochore-Bi-Attachment checkpoint. From Prometaphase to Metaphase: Motor driven chromosome congression Anaphase A and Anaphase B (from earlier to later Anaphase). Intermediate Filaments

2 Cytoskeleton Actin microfilaments are mainly responsible for the maintains of the cell shape, cell plasticity and the cell movement in many types of cells.

3 Microtubules Mitotic spindle

4 Tubulin dimer and its assembly in microtubules. Tubulin dimer consists of and tubulins bound in a polar manner: the head of TB to the tail of TB. The subunits are closely sequence and structure related being of about 55 kd. TB contains an un-exchangeable GTP which is never hydrolyzed whereas TB contains an exchangeable GTP which is readily hydrolyzed to GDP. The step along filament is equal to the size, 8 nm, of the tubulin dimer from the TB head of to the tail of TB. The minus (-) end of a protofilament contains TB whereas the plus end contains TB. Most microtubules contain 13 parallel proto-filaments assembled side by side into a tube-like structure. The real mode of the assembly is the addition of TB dimers to the plus end of the tubule in circular way so that the plus end grows, keeping with the tube geometry. In yeast there are two genes for TB and one for TB whereas several genes for TB and TB are present in higher multi-cellular eukaryotes. All 13-tubules have a seam where the TB subunit of the first filament meets the TB of the last, 13th filament. Otherwise, the TB subunit contacts the TB subunit in the neighboring filaments.

5 MT dynamics in vitro. The (+) end with TB subunits is the preferential end of the MT elongation. Here, the TB dimer binds. One can see that a special situation occurs when this circular binding passes the seam of the MT. Here the TB interacts laterally with TB. When a microtubule reaches a certain length (after it has passed the nucleation stage in vitro) the polymerization at the (+) starts when the TB dimer concentration exceeds a critical Cc(+). When the TB concentration is higher than Cc(-) then the MT will grow at both ends. If TB drops below Cc(+) then it will disassemble from both ends. An interning situation occurs when the TB concentration is higher than Cc(+) but lower than Cc(-). In this case the TB will depolymerase at its (- ) end and grow at its (+) end in a process called treadmilling. The general effect of treadmilling is an apparent MT movement. In vivo the concentration of TB in the cell may be higher than um while Cc is in range of 30 nm. This apparently tells us that MT should always polymerase in the cell from both ends. Fortunately, microtubule associated proteins (MAPs) regulate the polymerization process at both ends putting it under the control of the cell.

6 Dynamic instability of MTs. If one breaks an individual MT by applying shearing force into smaller ones some of them will grow but the others will shrink even at a TB concentration well above the critical one. The growing microtubules have blunt ends while the shrinking ones have horn-like structures at their ends containing separated microfilaments containing GDP- TB. At the growing (+) ends TB is preferentially in its GTP form (GTP- TB). Kinetics of dynamic instability. The shrinkage of microtubules is called the catastrophe while the growth is called the rescue. The microtubules emanating from the MTOC grow and shrink constantly showing dynamic instability unless they are captured by organelles or by other structures (like Kinetochores on chromosomes) in the cell. The capture would stabilize their (+) ends.

7 The reason for dynamic instability of microtubules is that the TB microfilaments containing GDP- TB are curved while those containing GTP- TB are straight. So, the MT filaments containing GDP- TB will be strained leading to dynamic instability since only the growing end will contain GTP- TB while inside the MT body we will have GDP- TB owing to GTP hydrolysis occurring soon after the TB dimer polymerization. The GTP-containing TB dimers at the (+) end have very strong lateral interaction between them, which prevents the pealing of GDP containing TB dimers inside the microfilaments in the bulk of the MT. The strain energy of GDP containing filaments in the tube can be used for fast MT disassembly at the (+) end when GTP in the capping (+) end is hydrolyzed or the cap is removed by a physical force as in vitro experiments.

8 MTOC. There are some variation in the number of filament in the microtubule which may also contain 11 or 15 filaments but most microtubules in the cell are singlets containing 13 filaments. The 13-MT is the only geometry where filaments are straight and not wind around the MT MTOC=Centrosome Most microtubules in the cell are nucleated from the structures known as microtubule-organizing centers (MTOC) with the (-) ends anchored at the MTOC. In interphase cells the MTOC is known as a centrosome, located near the nucleus. In mitotic cells there are two MTOCs that form Spindle poles. In interphase cells and neurons microtubules are used as rail-roads for vesicle and organelle transport. In mitotic cells they are used to separate chromosome towards MTOCs.

9 Centrosomes Centrosomes (MTOCs) in animals consist of a pair of orthogonally arranged cylindrical centrioles made of triplets surrounded by pericentriolar material. Centrioles are about 0.5 um long and 0.2 um in diameter. They are highly organized and stable structures that consist of nine sets of triplet microtubules. The factors in the pericentiolar material are instrumental in initiating microtubule nucleation. The main one is the TB ring complex. It acts as a template by bind the tubulin dimers at its plus end. TuRC is about 2.2 MDa. Colchicine drug binds to the tubulin dimer preventing Tb polymerization into microtubules. Taxol drug binds to microtubules stabilizing them and preventing their de-polymerization

10 TuRC in more details TuRC is assembled from several subunits. The main component is -tubulin that interacts with - Tb of the Tb dimer. Several additional components include Gcp2 and Gcp3 that can form Small Complex TuSC and several TuSC-like complexes containing Gcp4,5 and 6 subunits. Yeasts only have Gcp2 and 3. TuRC is assembled from several TuSC, and few TuSC like complexes in such a way that only 13 Tb are presented for template polymerization. The 14- Tb is hidden below the first one. In addition to centrosomes other structures in the cell can acquire and activate TuRC and initiate MT polymerization in the cell. Activation relies of conformational change in TuRC that brings Tb closer together making TuRC geometry to match that of the MT exactly. TuSC components are all phosphorylated by MPF and MPS1 kinases.

11 In yeast the ring complex assembles from TuSC complexes. Note that 14- Tb is hidden from the use in polymerization below the 1 st one. In high organisms Gcp 4,5 and 6 (gamma-complex protein) subunits are present. Additional proteins, like pericentrin, ninein, etc promote TuRC attachment and activation in the MTOC. XPAM215 promote fast growth at the + end in vivo by stabilizing Tb dimer binding to + ends. It uses its Tog domains to bind Tb dimer and other domains to bind MT. CLASP family prevent disassembly of MTs.

12 Microtubule associated proteins (MAPs). MAPs change the dynamics of MTs. Side-MAPs: These proteins have a modular design with one domain containing several repeats of 18 AAs, which are mainly positively charged and bind specifically to the negatively charged surface of MT formed by TB dimers. The other domain projects outside the MT regulating the spacing between adjacent parallel MTs. Tau is present mostly in axons of neurons to organize the axon processes while MAP2 is only found in dendrites. The lateral binding of positive domains of MAPs to negative MT surface stabilize MT preventing catastrophes. The protruding domains of Tau or MAP2 can be phosphorylated by specific protein kinases preventing their interaction with MTs which will promote MT disassembly. Tau is phosphorylated by MAPK/Par-1. Cyclin-CDK complexes can also phosphorylate MAPs regulating their association with MT during the cell cycle.

13 TIP-MAPs: +TIPs bind to the (+) end and either stabilize it or enhance the frequency of rescue..+tip protein EB1 binds near the (+) end in the seam stabilizing the growing (+) end. Disassembly of MTs Kinesin 13 binds to the (+) end and curves the TB dimer into GDP conformation. It also removes TB dimer from the (+) end removing the capping GTP containing TB dimers. Then Kin13 is released from the captured dimer upon ATP hydrolysis on Kin13 and it can repeat cycles of the (+) end pealing in a catalytic manner. This results in increased catastrophe frequency and MT shrinkage, which is important for chromosome separation in mitosis. Stathmin (Op18= Oncoprotein 18) binds two TB dimers forcing them into GDP conformation: this may promote GTP hydrolysis leading to the GDP cap TB pealing from the (+) end. Stathmin is regulated (inactivated) by phosphorylation. Katanin acts in MTOCs where it severs and releases anchored microtubules.

14 Kinesin motors. The structure of Kinesin 1 motor protein. Linker in the head domain has a primary importance for the motor movement along MT tracks Kinesins 1 and 2 participate mainly in vesicle transport. Kinesins 5 and 13 are important in mitosis.

15 Kinesin motors walk along MT using ATP energy.

16 Kinesin motors in the transport along microtubules. Kinesins are highly processive motors that can take several hundred steps on a microtubule without detaching from it. Step size 8 nm, Force 6 piconewtons, Speed 3 μm/s (25 cm/day). Different kinesins are either (+) end directed or ( ) end directed motors. Most kinesins, though, move towards the (+) end. Those have motor heads on their N-terminus. Kinesins can be divided also into transporting and mitotic kinesins, depending on the type of cargo they carry. The sequence of the unique tail domain and the presence of KLC (light chains) determine their cargo specificity.

17 Many kinesins are engaged in mitosis dealing with chromosome segregation/alignment and spindle organization, like its positioning and other functions in the spindle control.

18 Cargo transport by Kinesin motors Vesicle and cargo transport kinesins are activated by cargo binding whereas mitotic kinesins are activated/deactivated by phosphorylation. For Kinesine 1 (KIF5, for example), the absence of cargo results in a bent conformation of heavy chains which allows light chains to interact with the motor heads preventing ADP release and, hence, interaction with microtubules. The binding of its two cargo/adaptor proteins FEZ1 and JIP1 relives the inhibition by light chains and allows Kin1 to bind and step along the microtubule. Kinesin 7 (CENPE), mitotic kinesin, is activated by phosphorylation of its C-terminal tail on one site by MPS1 kinase present at a Kinetochore and on a different site by ClnB-CDK1 complex. Both phosphorylation are required to completely activate Kin7. This ensures that CENPE is only active when attached to the Kinetochore and only during mitosis when ClnB-CDK1 complex is active.

19 One example here is the use of Grip1 adaptor protein to connect KIF5 heavy chain with GluR2 repressor cytoplasmic domain

20 Binding and release of vesicles Vesicle can be transported by kinesins motors too. They may bind to the motors directly through their coat proteins. For example AP1 coat binds directly to KIF13 (Kin 3) through the 1 AP1 subunit. Another way to bind the vesicle cargo, like receptors (NMDA vesicle in this example), is to use protein adaptor complexes, like Lin complex. Cargo release can be also regulated by different signals. For example, Kin 2 (KIF17) transports its cargo-a vesicle packed with NMDA-R (N-Me- D-Asp)-Receptors. It can release the vesicle in response to neuron excitation in the vicinity of the vesicle. The excitation leads to Ca2++ influx that activates CamKII (calmodulindependent protein kinase II), which in turn phosphorylates the C-terminal cargo domain of Kin-2 which leads to its dissociation from LIN10 in the Lin scaffold complex and the cargo vesicle release.

21 Kinesin dependent transport in axon Motility of mitochondrion in neurons is regulated by Ca2+: KIF5 transports mitochondria using the Milton Miro complex as adaptor. Miro has two EF hand motifs that bind Ca2+ and sense intracellular Ca2+ levels. In response to high Ca2+ influx, these motifs bind Ca2+, change conformation and bind to the motor domain of KIF5. This leads to KIF5 detachment from the MT.

22 Rab-dependent vesicle attachment to kinesins Rab proteins that bind to vesicles can also be involved in the motor attachment. Here, KIF1A/B (Kin 3) binds to the synaptic vesicle when Rab is in GTP form. This Rab interacts with the adaptor protein DENN attached to the tail domain of KIF1A. The membrane binding capacity of PH (Pleckstrin Homology) domain of KIF1 is insufficient for tight binding. So, when RAB3 is inactivated by GTPaseactivating proteins at the axon terminus, the RAB3 GDP-carrying vesicle becomes released.

23 Dynein Motor. Cytoplasmic Dynein is a major versatile (-) motor in the cell cytoplasm that moves to the (-) end on the MT. Dynein uses ATP energy to produce a power stroke and it moves from one TB to the next TB along the MT filament with a step size 8 nm equal to the size of the TB dimer. Dynein complex is assembled from two identical heavy chains (>0.5 MDa each) that use N-terminal tail domain to bind a number of different subunits (LIC, IC, LC). Together, they form the core Dynein complex. Each heavy chains contains a C-terminal motor domain (head). Each head contains six AAA domains, four of which can bind and hydrolyze ATP. The tail provide scaffold for 5 different proteins: IC (Intermediate Chain) and LIC (Light IC) bind directly to Dynein tail. The other three proteins (two light chains, LS7 and LS8 plus TCTEX1) bind to the IC. P150 of Dynactin interacts with IC. During the movement one head remains attached while the other make a step, then the attached head detaches and steps while the other stays attached (alternative stepping, like in kinesin motors). This ensures the processivity of the motor.

24 Dynactin (from Dynein activator) itself is a multi-subunit complex that binds to Dynein through the p150 Glued subunit. Dynactin is required for Dynein activity in vivo. Dynactin complex (1 MDa) consist of two sub-complexes with p150 Glued sub-complex connected through the dynamitin subunit to the second sub-complex. The latter containing 8 actin related proteins (Arp1) caped by CapZ subunit on one end and by several other subunit at the opposite end. This sub-complex participates in cargo binding. The MT binding domain of dynactin increases Dynein processivity (providing an additional interaction with MTs). In addition to dynactin, another complex, LIS1-NUDE is generally required for the cellular function of Dynein. (LIS stays for the lissencephaly disease). Bicaudal D serves as another adaptor. Arp1 interacts with Spectrin.

25 Roles of Microtubules and Kinesins in Mitosis. In the S-phase the former mother centrioles generates the growth of the new daughter centrioles while the old daughter centriole becomes a new mother centriole and also generates the growth of a new daughter centriole. Starting from the middle of S-phase the new daughter centrioles grow and in G2-phase the pare of centrosomes separate and the new centriole becomes to acquire pericentriolar material. By G2 the growth of the (blue) daughter centrioles is completed and they start to move to the opposite poles of the cell each forming a spindle pole. This movement is directed to through the interaction of microtubules generated from centrosomes. As the cell enters mitosis the two MTOCs accumulate more and more pericentiolar material including the TuRC complexes, which greatly increases their ability to nucleate and radiate microtubules.

26 Mitotic Spindle: The MTs generated from the mitotic MTOC can be subdivided into three classes (they differ at their (+) ends, being modified by different attachments). The astral MT projects toward the cell wall cortex and position each spindle pole (MTOC) in the cell. The astral MTs orient the spindle with the axis of the cell division. MTs attached to Kinetochores. They bind to chromosome kinetochores on each of the sister chromosome and they are instrumental in positioning each chromosome pair in the division plane (metaphase plate) and in the pooling sister chromosomes to the opposite spindle poles in anaphase. Inter-Polar microtubules: they extend towards the opposite poles interacting together in the overlapping region in anti-parallel manner. These anti-parralel microtubules are connected by Kin5 which slide them relative one another.

27 The role of kinesin motors in mitosis. Kin 5 participates is the alignment of interpolar microtubule and in the positioning of centrosomes. In prophase Kin-12, 13 and 14 localize to centrosomes (MTOCs) where they function is spindle pole assembly and organization. Kin 7 (CENPA) and Kin 13 are localized to Kinetochore where they participate in Kinetochore MT capture while Kin 4 and Kin 10 localize to chromosome arms facilitating MT capture and chromosome congression. During anaphase Kin 7 and Kin 13 are the main kinesins which participate in kinetochore-mts dynamics, Kin-5 and 6 work mainly by sliding interpolar MTs.

28 Kin-10 positioning in chromosomal arms is promoted by Ran-GTP pathway Kin-10 is held inactive in complex (a/b) importin. High Ran-GTP concentration around chromatin releases Kin-10 in its vicinity and Kin-10 binds to chromosome arms. This high Ran:GTP concentration is due to the association of Ran GTP/GTP exchange protein (GEF) RCC1 with chromosomes during mitosis. MPF phosphorylate Kin 5 (Eg5) activating it for the binding to antiparallel interpolar microtubules where it participate in bipolar spindle formation. Phosphorylation of Kin 6 makes it inactive. In late anaphase Kin 6 is activated.

29 Complex structure of Kinetochore Kinetochore schematic structure and microtubule capture. The Kinetochore place on a chromosome is defined by positions of nucleosomes containing CENP-A and H2AZ histone variants. CENP-A is a specific variant of H3 histone. It differs from H3 around loop 1. In budding yeast a 125 bp DNA sequence determines centromere: CDEIII attract Ndc10 complex containing 3 subunits that assemble one CENP-A nucleosome over the ATrich element. Other higher eukaryotes may inherit centromeres epigenetically.

30 The centromeres seems to be folded in such a way that CENP-A nucleosomes emerge on one side of a sister chromosome. The simplest Kinetochore in SC yeast : CENPA nucleosome organizes all other complexes on Kinetochore. Dam1 ring attachment through the Nde80 complex to Kinetochore may explain how chromosomes segregation occurs in anaphase without motor proteins. Ipl1 complex is the AuroraB kinase.

31 From Prometaphase to Metaphase: Motor driven chromosome congression. Once MT attaches to a kinetochore the dynein/dynactin motor attached to kinetochore begins stepping towards MTOC to the (-) direction while Kin13 de-polymerase the MT leading to its shrinkage. The chromosome pair moves towards the attached MTOC to the right. This movement may also orients the unattached kinetochore of the sister chromosome towards the opposite pole allowing MTs radiating from this pole to get captured so that the two kinetochores of the chromosome pair become attached to the opposite spindle poles and become bi-oriented. The bi-attachment leads to the chromosome congression where the shrinking MT move the chromosome pair with the help of Dynein to the farthest pole while on the growing MT Kinesin 7 motor works in the (+) direction pushing the chromosome pair from the closest pole. In the growing MT the activity of dynein/dynactin motor is switched off and that of Kin 7 switched on. On the shrinking side dynein/dynactin is working while Kin7 is switched off. Kinesin 4 helps in positioning chromosomal arms at the metaphase plate.

32 From early to late Anaphase: Catastrophe driven segregation Important connections at Kinetochore: Anaphase: Ndc 80 and Dam1 complexes ensure the constant MT attachment to Kinetochores independent of the action of motor proteins. Anaphase A may proceed simply because MTs attached to Kinetochore depolymerase at their (+) end. A2: MT shrinks also at the (-) end due to Kin13 presence in the pole. B1: Kinesin 5 pushes poles apart. B2: Dynein anchored to the cell cortex pulls astral microtubules facilitating Anaphase B.

33 Intermediate Filaments (Ifs) Ifs are 10 nm wide (in between MTs and actin microfilaments) Ifs are very stable. Ifs polymerize spontaneously in vitro without the need for nucleotides or other cofactors IF proteins have no enzymatic activity Ifs are classified according to their occurrence in different tissues, and according to their sequence: Nuclear lamins Keratins in epithelial cells Vimentin in leukocytes, blood vessel endothelial cells, fibroblasts Neurofilaments (NFs) in axons of nerve cells. Two distinct types of intermediate filaments: lamins in the nuclear inner envelope and vimentin in the cytoplasm. Figure to the right: A: lamins. B: vimentin. C: DNA. D: Merged.

34 Similarity in organization for different Ifs. All Ifs can form similar protofibrils. Intermediate filament coil-coil dimers form side by side staggered tetramers which then form long protofilaments. Protofilaments assemble into protofibrils.

35 Arrangement of different filaments in the cell. Different filaments interact inside the cell combining their different properties.