12-8-08_MicrotubuleMotors_lectureC

12-8-08_MicrotubuleMotors_lectureC - Laying the tracks for...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: Laying the tracks for a moving train Mass transportation is doomed to failure in North America because a person's car is the only place where he can be alone and think. Marshall McLuhan In my district, the ports of Long Beach and Los Angeles handle approximately 44 percent of all of the goods delivered to American shores, yet they are in constant need of revenue for facilities, improvements and upgrades to roads and bridges and rails. Dana Rohrabacher Microtubules are made of tubulin dimers • α-tubulin and β-tubulin exist in dimers. – β-subunit has a bound GDP, which is exchanged for GTP during microtubule assembly – α-subunit has a bound GTP that is non-exchangeable • These dimers assemble in α-β-α-β-α-β chains called protofilaments. Microtubules are polarized and form helical cylinders • • • • • • • Staggering of adjacent protofilaments creates helix that is 3 subunits high/per full turn A seam is formed where alpha and beta subunits are directly adjacent to each other Protofilaments are asymmetric (α -tubulin at one end, β -tubulin at other), giving them a polarity. All protofilaments of microtubule have same polarity, called plus- & minus-end) Plus end - fast-growing (β -tubulins on tip); minus end - slow-growing (α -tubulins on tip) Polarity important for growth and transport Example: Axon, all plus-end distal. Dendrite, plus and minus-end distal Microtubule dynamics are regulated by microtubule associated proteins (MAPs) • • • • Mostly found in neural tissue, but MAP4 in non-neuronal cells One microtubule associating domain and one filament MAPs usually promote assembly and stability Association with microtubules controlled by phosphorylation Microtubule organizing centers • • • • Two stage formation of microtubules First is nucleation when initial portions of microtubule form Second is elongation In vitro, nucleation is slow. Made fast in cell by having microtubule organizing centers Best-studied is centrosome involved in organizing mitotic cell division Centrosomes nucleate microtubules • • Gamma-tubulin is necessary for microtubule nucleation • • • Gamma-tubulin colocalizes in all microtubule organizing centers Small proportion of total tubulin Gamma-subunits thought to bind to “ring complexes” of centrosome, which are the same diameter as microtubules Alpha-beta heterodimers (beta on plusend tip) can then bind to these gamma starter subunits • Assembly of microtubules requires GTP • • • • • GTP is bound to beta subunit of a soluble heterodimer GTP is hydrolyzed some time after dimer attaches to growing chain. GDP remains bound Plus-end of growing microtubule is more wide open and contains GTP dimers GTP on plus-end favors growth when possible Full conversion to GDP and closure of tip can lead to mechanical stress on protofilament and “catastrophic shrinkage” In vivo microtubule dynamics: Dynamic Instability • • • • • • Left: Biotin labeled tubulin after one minute in growing fibroblast. Tubulin incorporated widely at growing ends. Right: Fluorescence video of an individual microtubule labeled with fluorescent tubulin at low concentration (speckling) Gradual growth of microtubules followed by rapid shrinkage. Dynamic instability is the coexistence of growing and shrinking microtubules and rapid length change of individual microtubules Shrinkage and replacement occurs more quickly than growth in many conditions unless stabilized by MAPs (e.g. axons) Organelle microtubules are much more stable than cytoskeletal ones Motorin’, what’s your price for flight? Molecular motors on microtubules: kinesins and dyneins Motor proteins of cell - convert chemical energy stored in ATP into mechanical energy that is used to move cellular cargo attached to motor Why efficient cellular transport is a major concern Schematic of a motor neuron connecting to biceps muscle (30 cm long axon). From Peter Hollenbeck, Purdue University Structure of kinesin • • • • • Involved in anterograde transport of materials (towards plusend of microtubules) Tetramer consisting of two heavy chains and two light chains with a head, neck/stalk, tail structure Head is force generating side Tail is cargo binding side Best studied microtubule motor. Found in high concentration in axons and initially isolated from squid giant axon. Main model for kinesin movement along microtubule • • • • • A single kinesin molecule moves along a single MT protofilament (rate proportional to [ATP]; up to ~1 µm/sec) At low ATP concentrations, kinesins can be observed in distinct steps Each step is ~8 nm in length, which is the spacing between tubulin dimers along protofilament & requires hydrolysis of a single ATP molecule; moves 1 dimer at a time Motor protein tends to move long distances along individual MT without falling off (>1 µm) "Hand-over-hand" mechanism – the 2 heads alternate in taking leading & lagging positions; requires that trailing head swing 180° around leading head with each step. Currently, more evidence for this model. Video of hand over hand Kinesin mechanochemical cycle • • • • At the start of the cycle, the leading head (dark green) is bound to the microtubule. The trailing head (light green) is detached and bound to ADP. Binding of ATP to the front head causes a major conformational change. The rear head is thrown forward, past the binding site of the attached head, to another binding site further toward the plus end of the MT. Release of ADP from the second head (now in the front) and hydrolysis of ATP on the first head (now in the rear) brings the dimer back to the original state The two heads have switched their relative positions, and the motor protein has moved one dimer step along the microtubule protofilament. Alberts et al. Going retro: the dynein motor Binds to MTs Binds to cargo or dynactin • • • • • • First MT-associated motor found (1963). Responsible for moving cilia & flagella & was called dynein; Huge protein (~1.5 million daltons); made of 2 identical heavy chains & a variety of intermediate & light chains Each dynein heavy chain consists of large globular head with 2 elongated projections (~10X larger than a kinesin head); The head acts as a force-generating engine The stalk contains the MT-binding site situated at its tip The stem binds the intermediate & light chains, which are implicated in attachment to various types of cargo Minus-end directed movement of materials along microtubules • • Cytoplasmic dynein involved in positioning of spindle & chromosome movement during mitosis Also a minus-end-directed microtubular motor for positioning & movement of vesicles & organelles through cytoplasm In neurons, cytoplasmic dynein implicated in retrograde cytoplasmic movement (towards cell body from dendrites) Cytoplasmic dynein does not interact directly with membrane-bounded cargo, but requires an intervening multisubunit adaptor, dynactin • • Get your motor runnin’: Cilia and flagella • Left: Rapid wave-like motion of the flagellum of a sperm cell. Cell was photographed with stroboscopic illumination at 400 flashes per second. Waves of constant amplitude move continuously from the base to the tip of the flagellum. Right: beat of a cilium, which resembles the breast stroke in swimming. Fast power stroke followed by a slow recovery stroke. • • • Basal bodies are microtubule organizing centers for cilia and flagella • Basal bodies are at base of cilium/flagellum. MTs in these structures generated directly from basal body Basal bodies are identical in structure to centrioles; the two can even give rise to one another Sperm flagellum originates as a basal body derived from sperm centriole Conversely, sperm basal body typically becomes centriole during fertilized egg's first mitotic division • • • 9+2 structure Basal body/centriole structure Motor math: 9+2 structure of the axoneme • • Cilium core called an axoneme. It is an array of MTs that runs longitudinally through entire organelle Motile ciliary or flagellar axoneme, with rare exceptions, consists of 9 peripheral doublet MTs surrounding a central pair of single MTs; known as 9 + 2 pattern or array Peripheral doublets consist of 1 complete (A tubule; 13 subunits) MT & 1 incomplete (B tubule) MT with 10 or 11 subunits Highly conserved structure. This 9 + 2 pattern is seen in axonemes from protists to mammals. • • Sequence of events in ciliary/flagellar sliding motion • • • • • Dynein arms anchored along A MT of lower doublet attach to binding sites on B MT of upper doublet Dynein molecules undergo conformational change or power stroke; causes lower doublet to slide toward basal end of upper doublet Dynein arms detach from B tubule of upper doublet Dynein arms reattach to upper doublet to reset another cycle Sliding on one side of axoneme alternates with sliding on other side so part of cilium or flagellum bends first one way, then in opposite direction Regulating ciliary & flagellar locomotion - 2 factors important, Ca2+ & cAMP • Dynein power stroke • • • • • • Head ATP dependent cycle similar to kinesin ATP binding causes head conformation change and stem (tail) rotation Results in stalk rotating to + end, resulting in stem region moving relatively towards end ATP hydrolyzed Dynein returns to original conformation and detaches from tubule Dynein loses ADP. Stalk can bind to tubule again to restart cycle. Actin filament, and we do know why, ‘scuse me while I move this myosin • • • • Actin filament = microfilament = F-actin Actin is highly conserved, with 88% sequence identity between yeast and mammalian actin Actin critical for muscle contraction Also critical for non-muscle motility, such as phagocytosis, organelle movement, or growth cones, wound healing Actin is polarized like microtubules, having minus and plus ends Microfilaments are a series of actin subunits Actin forms double helices with grooves running along each side of helix when ATP present • • • Actin filament assembly and disassembly • • Before incorporation into a filament, an actin monomer binds a molecule of ATP (like tubulin binds GTP) Actin is an ATPase (like tubulin is GTPase); ATP role in MF assembly is same as GTP in MT assembly; some time after monomer incorporation into growing actin filament, its ATP is hydrolyzed to ADP Like tubulin, the filament end has an actin-ATP subunit cap, which hinders filament disassembly & favors continued assembly When MFs are incubated in vitro with a high concentration of labeled actin-ATP subunits, plus end incorporates monomers at 5 - 10 times higher rate than the minus end Minus (pointed) end is preferential depolymerization site so subunits treadmill Actin filaments exhibit dynamic instability similar to MTs. Dynamic instability = coexistence of randomly growing & shortening filaments • • • • Motörheads and motörtails of myosin II: involved in muscle contraction and non-muscle motility • All myosins share characteristic motor (head) domain, which has a site that binds the actin filament & one that binds & hydrolyzes ATP to drive the myosin motor • Tail domains are highly divergent between myosins • Myosins also contain a variety of low molecular weight (light) chains • Humans have ~40 different myosins, each presumed to have its own specialized function(s); Skeletal (striated) muscle cell structure is very unusual • • • • • • cylindrical cell; 10 - 100 µm thick; up to 40 mm long Skeletal muscle cells are multinucleate (hundreds of nuclei), Because of their properties, these cells are more appropriately called muscle fibers Muscle fiber is a cable made up of hundreds of thinner, cylindrical strands (myofibrils); seen in longitudinal section Each myofibril is made up of a repeating linear array of contractile units (sarcomeres) Each sarcomere, in turn, has characteristic banding pattern that gives muscle fiber a striated appearance Organization of vertebrate skeletal muscle Myofibrils are organized into repeating serial units called sarcomeres (the basic unit of contraction) Z disk (line) - network of proteins - thin filaments anchored here by α-actinin - area between two Z discs is a sarcomere M line – network of proteins - thick filaments anchored here Titan – filaments of huge elastin protein - runs from Z disk to M line helps restore sarcomere - anchored to M line and thick filaments length after stretch or contraction Myosin molecules spontaneously aggregate with tails overlapping and heads pointing outward Thick filaments with heads extending away The Sliding Filament Hypothesis for skeletal muscle contraction ● during contraction thin and thick filaments interact ● cross-bridges bind actin ● the thin filament is pulled along the thick filament toward the center of the sarcomere ● the bond is broken between the filaments and cross-bridge binds another site on actin ● the thin and thick filaments don’t change length ● degree of overlap between thin and thick filaments change Tropomyosin and the troponin complex associate with the actin thin filament Tropomyosin ● Filamentous protein that winds around actin filaments ●Blocks the myosin-binding site on actin in resting muscles Troponin complex Troponin C – binds Ca++ Troponin T – binds tropomyosin Troponin I – binds both tropomyosin and troponin C ●Troponin complex regulates tropomyosin position on actin The hydrolysis of ATP and the release of Pi change the conformation of the cross-bridge ● ATP hydrolysis energizes cross-bridge ● in ‘cocked’ position (neck) ● Release of Pi leads to release of energy ● head rotates relative to linker ● results in power stroke ● thin filament pulled past thick ● Binding of ATP to cross-bridge leads to detachment of thin and thick filaments ATP hydrolysis ...
View Full Document

This note was uploaded on 02/09/2010 for the course BIOL 230 taught by Professor Bartlett,e during the Fall '08 term at Purdue University-West Lafayette.

Ask a homework question - tutors are online