ScienceWeek

CELL BIOLOGY: ON MOLECULAR MOTORS

The following points are made by Roop Mallik and Steven P. Gross (Current Biology 2004 14:R971):

1) Cells are organized with different compartments -- the nucleus, the Golgi complex, the endoplasmic reticulum, and so on -- that act as factories. Each factory generates a unique set of products, which are then distributed to "consumers", which could be either end-users or other factories. The distribution system is complex, and uses three sets of molecular transporters: the myosin, kinesin, and dynein motors. Intracellular transport occurs along two sets of paths, both of which are similar to rail systems: the more or less randomly oriented actin filaments, used by myosin; and the (typically) radially organized microtubules used by both kinesin and dynein. Transport occurs along each of these when the appropriate motor binds to a cargo through its "tail" and simultaneously binds to the rail through one of its "heads". The motor then moves along the rail by using repeated cycles of coordinated binding and unbinding of its two heads, powered by energy derived from hydrolysis of ATP [1-4].

2) Microtubules are polar, and are typically organized with "minus ends" clustered at a microtubule-organizing center situated close to the nucleus. The microtubule "plus ends" spread outwards from the organizing center, and this leads to a radial organization of the microtubule network in some interphase cells, such as fibroblast cells [5], pigment cells, and certain mammalian cells. Microtubule organization is cell-type specific and in some cases, such as neurons and epithelial cells, differs significantly from the usual radial organization.

3) Motor proteins are able to recognize the microtubule polarity, and so the organization of the rails combined with the specific motor employed determines the direction of transport. Most kinesin-family motors that have been studied move toward the plus-end of the microtubules [1,2], and thus kinesin-mediated transport is usually used to bring cargos toward the cell periphery. In contrast, dynein moves in the other direction --toward the microtubule minus-end [1,2] -- and is typically used to move cargos toward the cell center (and nucleus).

4) Actin filaments are more randomly oriented, and can be used by unconventional myosin motors, such as myosin-V, to ferry cargos. Actin filaments are significantly shorter than microtubules and have been suggested to bridge the gap between microtubules, for example in cultured rat axons. In this way, local transport can occur on actin filaments in regions where there are few microtubules, as at the axon terminal. As with microtubules, the organization and density of actin filaments is cell-type specific. In some cases, actin filaments have an ordered structure close to the cell surface with barbed (plus) ends pointed outwards, which could allow myosin-V -- which moves toward the actin filament plus end -- to transport cargos to the very edge of the cell.

References (abridged):

1. Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519-526

2. Vale, R.D. (2003). The molecular motor toolbox for intracellular transport. Cell 112, 467-480

3. Vale, R.D. and Milligan, R.A. (2000). The way things move: looking under the hood of molecular motor proteins. Science 288, 88-95

4. Schliwa, M. and Woehlke, G. (2003). Molecular motors. Nature 422, 759-765

5. Rodionov, V., Nadezhdina, E. and Borisy, G. (1999). Centrosomal control of microtubule dynamics. Proc. Natl. Acad. Sci. USA 96, 115-120

Current Biology http://www.current-biology.com

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ON MYOSIN MOTOR PROTEINS

Notes by ScienceWeek:

Fifty years ago, a biologist looking at a large living biological cell through a light microscope could see motions on the surface and in the interior of the cell, motions aplenty and all of it mysterious. It was not until the 1960s that the microscale structures involved in cell movements were roughly identified, not until the 1970s that the biochemistry of these structures was characterized, and not until the 1990s that a clearer picture of the possible intricate movements of the "molecular motors" (motor proteins) of living cells became apparent.

An engineer viewing some of the current models of biological molecular motors will find nanoscale devices involving only a handful of macromolecules, with each device engaged in a precise sequence of repetitive movements -- rotations, vibrations, translocations along tracks, linear contractions, etc. -- the energy for these motions derived from enzyme-catalyzed reactions, and all of these devices assembled with apparent great precision by synthetic processes controlled by information stored in the genome of the cell. It is quite understandable if the engineer, for example, while looking at a model of the macromolecular assembly evidently responsible for the rotation of a flagellum, the whip-like structure involved in bacterial movement, is flabbergasted. We have apparently crossed a threshold into a world of nanoscale "machinery" in biological cells, and cell biology in the 21st century promises to be a source of extraordinary revelations.

It is now recognized that the interiors of biological cells are structurally complex, and that this structure is dynamic. Microtubules are part of the cytoskeleton of biological cells, the quasi-rigid matrix that among other things determines cell shape. The microtubules are 25 nanometers in diameter, and composed of the protein tubulin. They occur in regular arrays in various cell organelles, and in the cytoplasm in general, and they contribute not only to cell shape, but also to cell motility.

Microfilaments are 4 to 6 nanometers in diameter, highly variable in length, and are found in all eukaryotic cells. They are composed of a protein called "actin" and several other accessory proteins, and they are important in cell locomotion and in the molecular dynamics of muscle cells.

"Motor proteins" are mechanico-chemical enzymes involved in locomotion or transport, and there are three families of such proteins: kinesins, dyneins, and myosins. Kinesins and dyneins are microtubule based motor proteins, while myosin is a microfilament based motor protein. In general, as mechanico-chemical enzymes, motor proteins convert energy from hydrolysis of nucleotides to mechanical force, and since they are involved in many important cellular events, the molecular details are currently the focus of intensive research.

Myosin is a large protein with a molecular weight of approximately 500K daltons, and it accounts for approximately half the protein present in the myofibrils that comprise muscle fibers. The myosin molecule consists of 6 polypeptide subunits: 2 heavy chains with a molecular weight of approximately 200K daltons each and 4 light chains of approximately 20K daltons each. In electron micrographs, purified myosin appears as a long thin rod containing 2 globular heads protruding at one end. This 2-headed type of myosin is called "myosin-2" to distinguish it from the smaller and single-headed myosin-1 molecule involved in cytoplasmic movements in some non-muscle cells.

The following points are made by Michael A. Geeves (Nature 2002 415:129):

1) Most organisms, whether they consist of a single cell or billions, can move in a directed way, an ability that is largely attributed to molecular motor proteins. Of these, myosin-2 is perhaps the best understood because of its role in muscle contraction. But other motors from the myosin family are also required for processes involving motility, from cell division to the transport of organelles within cells (1). One of the most hotly debated issues (2) in this field centers on how myosins move, and a theory known as the "lever-arm hypothesis" has received much experimental support. This theory proposes that tiny changes in myosin's "head" portion are amplified by the adjoining "neck" (the lever arm) to produce large displacements at the far end of the neck that translate into movement of the whole protein (3), with the size of the displacement depending on the length of the lever. Not everyone agrees, however, and the theory has faced recent challenges.

2) Myosins consist of a head, a neck and a tail. The neck comprises a structural element identified as an alpha-helix, often attached to up to six polypeptide chains called light chains. The tail is involved in connecting the motor to its cargo, specifying the motor's cellular location and, in some cases, allowing dimerization. (Myosins 2, 5 and 6, for example, all consist of two identical proteins, each with its own head, neck and tail.) The head attaches to and moves along tracks of actin filaments. These are made up of globular monomers that string together to form a chain; two chains twist round each other to form a helical filament.

3) The breakdown of the cell's energy store, adenosine triphosphate (ATP), powers myosin movement, driving large structural changes that cause the myosin heads to cyclically attach and detach from actin. Crystal structures of the myosin II head show the neck emerging from it, stabilized by two light chains, at an angle that varies by up to 60 degrees depending on whether the head is bound to ATP or to the products of ATP hydrolysis (adenosine diphosphate and phosphate). So the neck looks like a lever, which led to the idea that it operates as a rigid body to amplify small structural changes in the myosin head, with longer necks leading to a larger displacement. Support for this model comes from studies of myosins engineered to have lever arms of different length. The results show a linear relationship between the lever's length and the protein's speed of moving actin filaments over a surface or step size in an optical trap.

References (abridged):

1. http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html

2. Cyranoski, D. Nature 408, 764-766 (2000)

3. Geeves, M. A. & Holmes, K. C. Annu. Rev. Biochem. 68, 687-728 (1999)

Nature http://www.nature.com/nature

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MOLECULAR BIOLOGY: ON THE MOTOR PROTEIN KINESIN

The following points are made by A. Yildiz et al (Science 2004 303:676):

1) Conventional kinesin (referred to simply as kinesin) is a highly processive, dimeric motor that takes 8.3-nm steps along microtubules (1-3). Kinesin transports a variety of cargo, including membranous organelles, mRNA, intermediate filaments, and signaling molecules (4). Mutations in a neuron-specific conventional kinesin have been linked to neurological diseases in humans (5).

2) Kinesin is a homodimer with identical catalytic cores (heads) that bind to microtubules and adenosine triphosphate (ATP). Each head is connected to a "neck-linker", a mechanical element that undergoes nucleotide-dependent conformational changes that enable motor stepping. The neck linker is in turn connected to a coiled coil that leads to the cargo-binding domain. In order to take many consecutive steps along the microtubule without dissociating, the two heads must operate in a coordinated manner, but the mechanism has been controversial. Two models have been postulated: the hand-over-hand "walking" model in which the two heads alternate in the lead, and an inchworm model in which one head always leads.

3) The hand-over-hand model predicts that for each ATP hydrolyzed the rear head moves twice the center of mass, whereas the front head does not translate. For a single dye on one head of kinesin, this leads to a prediction of alternating 16.6-nm and 0-nm translation of the dye. In contrast, the inchworm model predicts a uniform translation of 8.3 nm for all parts of the motor, which is equal to the center-of-mass translation. In addition, each model makes predictions about rotation of the stalk. The inchworm model predicts that the stalk does not rotate during a step. A symmetric version of the hand-over-hand model, in which the kinesin-microtubule complex is structurally identical at the beginning of each ATP cycle, predicts that the stalk rotates 180 degrees, whereas an asymmetric hand-over-hand model does not require stalk rotation. Based on biophysical measurements that showed no rotation of the stalk, Hua et al (2002) concluded that an inchworm model was more likely for kinesin, although they could not rule out an asymmetric hand-over-hand mechanism.

4) In summary: Kinesin is a processive motor that takes 8.3-nm center-of-mass steps along microtubules for each adenosine triphosphate hydrolyzed. Whether kinesin moves by a "hand-over-hand" or an "inchworm" model has been controversial. The authors have labeled a single head of the kinesin dimer with a Cy3 fluorophore and localized the position of the dye to within 2 nm before and after a step. The authors observed that single kinesin heads take steps of 17.3 +- 3.3 nm. A kinetic analysis of the dwell times between steps shows that the 17-nm steps alternate with 0-nm steps. These results strongly support a hand-over-hand mechanism, and not an inchworm mechanism. In addition, the authors suggest their results indicate that kinesin is bound by both heads to the microtubule while it waits for adenosine triphosphate in between steps.

References (abridged):

1. J. Howard, A. J. Hudspeth, R. D. Vale, Nature 342, 154 (1989)

2. K. Svoboda, C. F. Schmidt, B. J. Schnapp, S. M. Block, Nature 365, 721 (1993)

3. R. D. Vale, R. A. Milligan, Science 288, 88 (2000)

4. L. S. Goldstein, A. V. Philp, Annu. Rev. Cell Dev. Biol. 15, 141 (1999)

5. E. Reid et al., Am. J. Hum. Genet. 71, 1189 (2002)

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