Lecture 19: Microtubule Motors 2024 PDF

In most cells, microtubules do not grow randomly; instead they grow from a nucleating organelle called a centrosome Centrosomes are composed of two parts: 1. A central pair of centrioles that are barrel-shaped organelles that are themselves made of bundled triplet microtubules 2. A...

In most cells, microtubules do not grow randomly; instead they grow from a nucleating organelle called a centrosome Centrosomes are composed of two parts: 1. A central pair of centrioles that are barrel-shaped organelles that are themselves made of bundled triplet microtubules 2. A surrounding matrix of proteins (visible as an electron  dense “cloud” surrounding the centrioles in the micrograph on the left) called the pericentriolar material Centrioles act as a scaffold for the pericentriolar material The pericentriolar material is made up of numerous proteins and is the site of microtubule nucleation This video shows an experiment in which a centrosome was purified from cells and attached to a microscope coverslip to be viewed by time-lapse fluorescence microscopy Fluorescent tubulin and GTP were then added and microtubules can be observed to grow out from the centrosome like a seed Once they start to grow, the  microtubules exhibit dynamic instability, growing and shrinking Not that new microtubules only grow from the centrosome – it is acting as a nucleation site in this purified system just as it would inside a cell Microtubule polymerization from centrosomes requires another type of tubulin: γ-tubulin γ-tubulin in the pericentriolar material acts as the seed for new microtubule nucleation This arrangement leads to a radial organization of the microtubule network with the fast growing plus end oriented out into the cytoplasm The minus ends – are capped by their interaction with γ-tubulin and anchored at the centrosome in most cells Microtubule polymerization from centrosomes requires another type of tubulin: γ-tubulin γ-tubulin in the pericentriolar material acts as the seed for new microtubule nucleation This arrangement leads to a radial organization of the microtubule network with the fast growing plus end oriented out into the cytoplasm The minus ends – are capped by their interaction with γ-tubulin and anchored at the centrosome in most cells In this lecture, we will investigate microtubule motor proteins and their regulators. Video on the left was made using enhanced light microscopy to image organelle motility in extruded cytoplasm The image on the right was generated with TEM to image organelle-microtubule interactions Kinesins are a family of motor proteins that are usually homodimers Kinesins have a head domain that binds to microtubules and to ATP and a tail domain that attaches to the cargo that they are transporting These cargoes can be vesicles, organelles, mRNAs, or protein complexes Kinesins walk along  microtubules towards the plus ends Dyneins also have globular head domains that are a bit different from kinesins (as we will see) and also have tails that attach to cargoes Dyneins walk towards the microtubule minus end BOTH motors hydrolyze ATP to produce the conformational changes that power their coordinated stepping movements along the microtubule 15 Most kinesins form homodimers. Each chain usually has an N- terminal motor domain that binds and hydrolyzes ATP, and this ATPase cycle underlies a mechanochemical cycle of binding and releasing from a microtubule. To do this, at least one of the motor domains must be bound to a microtubule at any one time, thus, the heads are coordinated in the ATPase cycles. 16 Here we will discuss three types of in vitro assays used to gain information on a motor’s activity: what is the speed that the motor moves along a microtubule? How long does a motor move on a microtubule before it dissociates? Does a motor move to the plus end or the minus end of a microtubule? What size step does a motor take per ATP hydrolyzed? These are the types of questions that can be answered with these assays. Some assays involve bulk motor action (many motors operating on a single microtubule) while other assays look at the action of a single motor (usually a homodimer). 17 Two prime assays to look at microtubule motor-based motility. A) above, microtubules serve as tracks in a microscopy assay – along which motors travel. Here, the readout for motility is the visualization of a vesicle as it moves. B) Below, this experiment changes the laboratory frame: Microtubule motors are fixed on a slide (effectively, the coverslip becomes their cargo). Microtubules are then flowed into the chamber and the motors “walk” along the microtubule. Since the motors cannot move relative to the slide they are fixed on, the microtubule moves instead, often with multiple motors attached along its length. If you have a microtubule with its polarity marked, a kinesin will cause the microtubule to move, leading with its minus end. In contrast, a dynein will cause the microtubule to move, leading with its plus end. In this assay, the experimenter monitors the movement of the microtubule, measuring which direction it moves (leading with it plus or minus end) and what velocity it travels at, which informs motor direction and speed respectively. Also shown in this assay is a bead with a motor bound to it, attached to a microtubule. This is a separate assay in which bead motility along a microtubule can be monitored (again, measuring directionality of the bead along a polarity marked microtubule, and the speed of the bead along a microtubule). For bead assays, the microtubules are usually fixed to the coverslip. 18 Here, kinesin is fixed to a slide, and microtubules are moved by the motors they are attached to. The microtubule seed is brightly labeled. The microtubule that polymerized off the seed is dimly labeled with fluorophore. Since the plus end polymerizes faster than the minus end, we can determine that the end of the dimly labeled microtubule region in the plus end of the microtubule. The microtubule moves, leading with its minus end, which means that the motors attached to the coverslip are plus end directed motors. 19 20 The ATP hydrolysis cycle of one kinesin motor domain regulates its binding affinity for the microtubule, as well as how it positions the other motor domain that is part of the dimer. Note that when the microtubule-bound kinesin motor domain transitions from the nucleotide free state to the ATP-bound state, it repositions the other kinesin motor domain from behind (more “minus-end”) to in front (more “plus-end”). The relative positional interplay between the two motor heads, and the out-of-phase coupling of their respective ATP hydrolysis cycles underlies the directionality of movement, and how processive the motor is (how many steps the motor takes before falling off the microtubule). 21 The ATP hydrolysis cycles of the each motor domain in the homodimer are coordinated, enabling coordinate bind-and- release, out of phase of one another – just like a persons legs alternate in touching the ground and moving forward when walking. 22 The kinesin family is large. You have many genes for kinesin motors. Most are homodimers, others are tetramers. Most are plus end directed. The NCD family is a unique microtubule minus end directed kinesin motor (you have these, but plants specifically rely on them because they lack dynein motors). Other kinesins bind to microtubule ends and depolymerize them (use motor activity to bind and depolymerize the microtubule). Many of these kinesin motors have accessory light chains that enable cargo binding specificity. Dynein has a motor region consisting of a ATPase domain (binds 4 ATP molecules), and a microtubule binding domain. Note that the microtubule binding domain is distal to the ATPase domain. ATP hydrolysis is going to cause a conformational change in the microtubule binding domain by sliding the two alpha helices that connect the two regions in opposite directions. This will result in microtubule binding and release. Shown is just a monomer of the motor head. But note that cytoplasmic dynein (like conventional kinesin) is a homodimer – and has two motor domains that are coordinated to walk hand over hand. Dynein is bound to the microtubule (MT) in the nucleotide free state (top) ATP binding causes the head to release from the MT (not shown, the other dynein head stays bound to the MT) – but positions the released head forward. ATP hydrolysis leads to the ADP-Pi state, this conformation has high MT binding affinity. The Pi is released, generating a power stroke – the magenta linker region moves (not shown, this would position the rear head in a forward position – more towards the minus end). ADP release completes the circle, but dynein is one step forward now – towards the microtubule minus end. Although dynein was discovered in the 1960s, we are still learning how dynein walks at the molecular level– an area of intense focus for cell biologists This is a model for how we think it moves. While kinesin takes 8 nm steps along a single protofilament towards the plus end, dynein can take a range of steps: 8, 16, 32 nm, and it can move from one protofilament to another as it walks along the microtubule. Dynactin is a key adaptor for cytoplasmic dynein: it links dynein to cargoes. Key dynactin components are shown in blue, above. Dynactin attaches to specific sets of factors that help mediate the interaction with dynein homodimers. Shown above are the proteins BICDR-A and –B. These specific adaptors recruit two dynein homodimers. Others recruit only one dynein homodimer. Collectively – specific dynein-dynactin complexes are used to recruit dynein to specific cargoes, and activate the motors so that cargo is transported. When dynein homodimers are not bound to dynactin, the motor adopts an autoinhibited state – this prevents the motor from moving along microtubules if it has no cargo attached. 27 In many species, microtubules have a net orientation in the neurons, or orientation in a specific array (e.g. mitotic array). Along these polarized tracks, cargoes are transported in specific directions. Some cargoes will have both motor types attached, and one will be specifically inactivated, or the number of one motor type dominates the net force, leading to directed transport. Cilia use IFT trains to transport cargo in the diffusion-limited space of the cillium. Kinesins bring cargo or “trains” to the cilia tip (plus end) in a process called anterograde transport, while dynein brings the trains back to the cilium base (retrograde transport). Each transport uses different tubules so that there are no collisions. As the trains have both dyneins and kinesins attached, the dyneins are inactivated during anterograde transport, while the kinesins are inactivated during the retrograde transport. One of the major functions of microtubules is to act as tracks for organelle positioning The ER (blue) and Golgi apparatus (yellow) are positioned within the cell by their interactions with the microtubule network ER tubules are pulled out along microtubules to reach into the cell periphery The Golgi is held in a position  next to the nucleus by interactions with microtubules These localizations are mediated by motor proteins that walk along microtubules 29 An example of bidirectional microtubule transport: pigment granule movement. 30 Different numbers of dynein or kinesin can be attached to a scaffold in vitro, and the motility of that complex monitored over time as it moves along a microtubule. The movement can be analyzed by kymography, just as we used kymographs to examine microtubule dynamics. From this, we can ascertain directionality of movement, speed, how long a complex is associated with a microtubule, and how often it pauses, or switches direction (if ever). Flagella and motile cilia are motile structures used to power cell motility (in cells such as sperm, left) or to move fluid over the surfaces of cells (as in the TEM on the right showing the surface of epithelial cells that line the respiratory tract). [Note that there are two types of cilia: motile cilia and non-motile cilia. The non- motile cilia are sensory cilia (such as the rods and cones in your eye) – and are used for sensory detection. The non-motile cilia do not have axonemal dyneins, though they do have cytoplasmic dyneins involved in IFT.] Cilia and flagella possess a core of microtubules arranged into a structure called an axoneme (shown in cross-section, center) Cilia and flagella have similar internal structures, they are distinguished by their number and length Flagella are long and present in 1 or 2 copies per cell Motile cilia are much shorter and present in 10-100 copies per cell They both drive cellular movements and their motility is powered by axonemal dyneins Motile cilia line the epithelial tissue of the respiratory tract to sweep particulate matter out of the airways  Motile cilia line the oviduct to push the egg Flagella allow sperm to swim This video shows the surface of a biopsy of tracheal cells taken from the respiratory tract of a mouse Tracheal cells are epithelial cells; their apical domains are overed in cilia These cilia line the respiratory tract to catch particles that we breathe and sweep them back towards the head to eliminate them (e.g. by coughing) Note how these tufts of cilia beat in synchrony to perform their filtration function Cilia beat by cycling through power strokes and recovery strokes. Axonemal dyneins are the molecular motors that drive this process. During the power stroke the cilium (or flagellum) is fully extended to fluid over the surface of the cell During the recovery stroke the cilium curls back to minimize disturbance of the surrounding extracellular fluid Each cycle occurs in ~1/10th of a second  The forces produced by the power stroke are parallel to the surface of the cell The axoneme is the core microtubule-based structure within cilia and flagella Left shows a TEM section through a flagellum, right is a cartoon structure depicting organization of the proteins Microtubules are arranged in a “9+2” array, 9 doublet outer microtubules surround 2 singles microtubules Each doublet microtubule carries  two rows of axonemal dyneins with each dynein extending its motor domain to interact with the neighboring doublet microtubule These dyneins attempt to walk along the neighboring doublet microtubules thereby producing the force that drives ciliary beating The microtubule bundles are held in place by radial spoke proteins and cross-linkers called nexins that help to convert the forces produced by dynein into bending of the axoneme If the doublet microtubules are purified from axonemes and ATP is added, then the doublets slide against one another and telescope away as the dyneins walk In the intact flagellum, the doublets are attached to one another by the cross-linking proteins (e.g. nexin). Instead of sliding the doublet microtubules continuously relative to one another, dynein motility is  converted to bending Repeated cycles of bending produce flagellar (or ciliary) beating.