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ScienceWeek
NEUROBIOLOGY: ON MICROTUBULE TRANSPORT IN GROWING AXONS
The following points are made by Y. Ma et al (Current Biology 2004 14:725):
1) In neurons, tubulin is synthesized primarily in the cell body [1], whereas the molecular machinery for neurite (axon) extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon.
2) According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment [2], it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods.
3) Tubulin can be delivered to the axon from the cell body either in the form of microtubules (MTs) or in the form of dimers/short oligomers. Initially it had been proposed that tubulin was transported in the form of crosslinked MTs ("coherent transport model" [3,4]). This idea, however, has been firmly rejected as a result of photobleaching [5], photoactivation, and fluorescence speckle microscopy experiments. Recently, it has been found that neurofilaments can be transported along the axon in assembled form. The movements were rapid, transient, and interrupted by prolonged pauses. The observed translocation of neurofilaments has led to the suggestion that rapid and asynchronous movement of MTs may be the basis of slow axonal transport of tubulin [4]. In agreement with this idea, rapid, intermittent transport of short (1-4 microns) tubule-like structures containing tubulin has been detected in the axons of sympathetic neurons, and it has been suggested that these structures represent individual MTs.
4) The authors measured the translocation of MT-plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. The authors suggest their results indicate that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.
References (abridged):
1. Campenot, R.B. and Eng, H. (2000). Protein synthesis in axons and its possible functions. J. Neurocytol. 29, 793-798
2. Okabe, S. and Hirokawa, N. (1990). Turnover of fluorescent-labelled tubulin and actin in the axon. Nature 343, 479-482
3. Black, M.M. and Lasek, R.J. (1980). Slow components of axonal transport: two cytoskeletal networks. J. Cell Biol. 86, 616-623
4. Shah, J.V. and Cleveland, D.W. (2002). Slow axonal transport: fast motors in the slow lane. Curr. Opin. Cell Biol. 14, 58-62
5. Chang, S., Girod, R., Morimoto, T., O'Donoghue, M., and Popov, S. (1998). Constitutive secretion of exogenous neurotransmitter by nonneuronal cells: implications for neuronal secretion. Biophys. J. 75, 1354-1364
Current Biology http://www.current-biology.com
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CELL BIOLOGY: ON MICROTUBULES
The following points are made by A. Dammermann et al (Current Biology 2003 13:R614):
1) Microtubule arrays direct intracellular organization and help to define cellular morphology. The cellular functions of microtubules critically depend on their intrinsic polarity, which results from the head-to-tail association of the alpha/beta tubulin subunits. This polarity is central to the ability of motor proteins to move unidirectionally on the polymer lattice and execute their diverse functions [1]. Microtubule polarity is also reflected in the distinct dynamic properties of the two polymer ends. Based on the analysis of microtubules assembled from purified tubulin, the faster polymerizing end was termed the "plus" end and the more slowly polymerizing end the "minus" end. Microtubule plus ends and proteins that affect their behavior have been the subject of much recent attention [2,3].
2) Microtubule arrays in cells are generally portrayed with dynamic plus ends exploring the cytoplasm and inert minus ends anchored at microtubule organizing centers. However, direct observation of microtubules in a variety of cell types has shown that the fraction of anchored minus ends varies extensively, from essentially all to practically none. Within organized arrays, the minus ends of microtubules can also be dynamic, as is the case for spindle microtubules in metazoans[4]. Microtubules with free minus ends may be generated by release from a microtubule organizing center [5], cytoplasmic assembly, or breakage/severing of existing microtubules. In different cellular contexts, each of these mechanisms is thought to make an important contribution to the steady state nature of the cellular microtubule array.
3) An interesting example of an array with a majority of unanchored microtubules is found in the lamellae of migrating epithelial cells, where 80-90% of microtubules have free minus ends. In this subcellular domain, spontaneous nucleation and centrosomal release contribute only negligibly to microtubule number. Instead, the majority of new microtubules arise via breakage of existing microtubules in a "convergence" zone where the retrograde flow of filamentous actin and microtubules from the lamellum collides with the slower anterograde flow of filamentous actin from the cell body. This effectively couples generation and turnover of microtubules to the dynamics of the actin cytoskeleton. A strikingly similar behavior has been observed in the migrating growth cones of neurons, suggesting that this mechanism may have general relevance.
4) In summary: Microtubules are polymers of tubulin, which is itself a heterodimer composed of alpha- and beta-tubulin. During microtubule polymerization, the head-to-tail association of alpha/beta-tubulin heterodimers results in linear protofilaments. Within the microtubule, thirteen protofilaments associate laterally to form a hollow cylindrical structure that measures 25 nm in diameter. Heterodimers are oriented within the polymer lattice with beta-tubulin pointing towards the faster polymerizing "plus" end, and alpha-tubulin pointing towards the slower polymerizing "minus" end. Alpha- and beta-tubulin are highly related proteins, ~50% identical at the amino acid level, and both bind GTP. When tubulin dimers polymerize, the GTP bound to beta-tubulin is hydrolyzed, and the resulting GDP does not exchange as long as the heterodimer remains in the polymer lattice. In contrast, alpha-tubulin binds GTP in a non-exchangeable manner and does not hydrolyze its bound nucleotide during polymerization. The uniform orientation of tubulin heterodimers in the microtubule lattice confers a polarity that plays a crucial role in the functions of the microtubule cytoskeleton.
References (abridged):
1 Goldstein, L.S. and Philp, A.V. (1999). The road less traveled: emerging principles of kinesin motor utilization. Annu. Rev. Cell Dev. Biol. 15, 141-183
2 Carvalho, P., Tirnauer, J.S., and Pellman, D. (2003). Surfing on microtubule ends. Trends Cell Biol. 13, 229-237
3 Howard, J. and Hyman, A.A. (2003). Dynamics and mechanics of the microtubule plus end. Nature 422, 753-758
4 Wittmann, T., Hyman, A., and Desai, A. (2001). The spindle: a dynamic assembly of microtubules and motors. Nat. Cell Biol. 3, E28-E34
5 Abal, M., Piel, M., Bouckson-Castaing, V., Mogensen, M., Sibarita, J.B., and Bornens, M. (2002). Microtubule release from the centrosome in migrating cells. J. Cell Biol. 159, 731-737
Current Biology http://www.current-biology.com
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Related Material:
CELL BIOLOGY: MICROTUBULES
Notes by ScienceWeek
The structural framework of eukaryotic cells (biological cells with nuclei and other membrane-bound internal structures), called the "cytoskeleton", consists of an arrangement of macromolecular structures: microtubules, intermediate filaments, and microfilaments. The microtubules are hollow cylinders approximately 24 nanometers in diameter, many microns in length, and consist of heterodimers of alpha- and beta-tubulin proteins plus a variable set of other proteins. They form the scaffolding of the mitotic spindle (an important structure in cell division), organize other cytoplasmic structures, and are the structural core of various organelles involved in cell movement (cilia and flagella).
Beginning in the early 1960s, the work of S. Inouye provided evidence that microtubules exist in equilibrium with free tubulin: microtubule assembly-disassembly is regulated by factors that influence the equilibrium between polymerized and nonpolymerized tubulin. In the early 1970s, R. Weisenberg demonstrated that microtubules assemble spontaneously in cell extracts that have been warmed to 37 degrees centigrade in the absence of calcium ions and in the presence of guanosine triphosphate.
In general, under appropriate conditions, microtubules spontaneously assemble when a solution containing purified tubulin protein and guanosine triphosphate is warmed from 7 degrees centigrade to 36 degrees centigrade. In the laboratory, following the spontaneous assembly of tubulin heterodimers into microtubules, the microtubules further spontaneously organize into superstructures, striped or circular macroscopic patterns that can be detected with suitable polarization optics. These macroscopic patterns are formed as a connected final self-organization following tubulin polymerization into microtubules. According to J. Taboy et al (1990), these macroscopic microtubule patterns are evidence of a self-organizing system behaving according to "reaction-diffusion mechanisms".
The following points are made by Joe Howard and Anthony A. Hyman (Nature 2003 422:753):
1) The textbook functions of microtubules are to act as beams that provide mechanical support for the shape of cells, and as tracks along which molecular motors move organelles from one part of the cell to another. To perform these functions, a cell must control the assembly and orientation of its microtubule cytoskeleton. Microtubules assemble by polymerization of alpha-beta dimers of tubulin. Polymerization is a polar process that reflects the polarity of the tubulin dimer, which in turn dictates the polarity of the microtubule. In vitro, purified tubulin polymerizes more quickly from the "plus" end, which is terminated by the beta-subunit. The other, slow-growing end is known as the "minus" end, and is terminated by the alpha-subunit.
2) In animal cells, minus ends are generally anchored at "centrosomes", which consist of specialized microtubule-based structures called "centrioles", surrounded by pericentriolar proteins. In yeast, the analogous structure is the spindle pole body. An important component of the centrosome is an unusual form of tubulin, gamma-tubulin, which is thought to initiate nucleation by forming rings that act as templates for new microtubule growth. After nucleation, microtubules grow out with their plus ends leading into the cytoplasm. Thus to a first approximation, the distribution of the microtubule cytoskeleton is determined by the location of the centrosome.
3) The first clue as to how cells rearrange the distribution of microtubules came from the discovery that during the polymerization of pure tubulin, plus ends switch between phases of slow growth and rapid shrinkage. The conversion from growing to shrinking is called "catastrophe", whereas the conversion from shrinking to growing is called "rescue". Analysis in tissue culture cells and in cellular extracts soon confirmed that this behavior, termed "dynamic instability", is a feature of microtubules growing under physiological conditions.
4) In summary: An important function of microtubules is to move cellular structures such as chromosomes, mitotic spindles and other organelles around the inside of cells. This is achieved by attaching the ends of microtubules to cellular structures; as the microtubules grow and shrink, the structures are pushed or pulled around the cell. How do the ends of microtubules couple to cellular structures, and how does this coupling regulate the stability and distribution of the microtubules? It is now clear that there are at least three properties of a microtubule end: it has alternate structures; it has a biochemical transition defined by GTP hydrolysis; and it forms a distinct target for the binding of specific proteins. These different properties can be unified by thinking of the microtubule as a molecular machine, which switches between growing and shrinking modes. Each mode is associated with a specific end structure on which end-binding proteins can assemble to modulate dynamics and couple the dynamic properties of microtubules to the movement of cellular structures.
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