Microtubules MAPs

MAP is the acronym for both, Microtubule Associated Protein and Mitogen Activated Protein. 

Microtubule associated proteins (MAPs) are attached to microtubules in vivo and play a role in their nucleation, growth, shrinkage, stabilization and motion. 

The cytoskeleton also contains different accessory proteins, which, in accordance with their affinities and functions, are designated as microtubule-associated proteins (MAPs), actin-binding proteins (ABPs), intermediate-filament-associated proteins (IFAPs), and myosin-binding proteins. This chapter is focused on those parts of the cytoskeleton that are composed of microfilaments and microtubules and their associated proteins. The subject of intermediate filaments is dealt with in detail in Volume 2. 

Microtubules can be reconstituted in vitro at 37 °C from a solution that contains a physiological mixture of brain tubulin, MAPs, small amounts of guanosine 5 -triphosphate (GTP), magnesium ions, and the calcium-chelating agent EGTA [ethylene glycol-bis(2-aminoethyl ether) N, N -tetraacetic acid]. Tubulin assembly is inhibited by low temperature and by the presence of calcium ions. 

Microtubule-associated proteins bind to microtubules in vivo and subserve a number of functions including the promotion of microtubule assembly and bundling, chemomechanical force generation, and the attachment of microtubules to transport vesicles and organelles (Olmsted, 1986). Tubulin purified from brain tissue by repeated polymerization-depolymerization contains up to 20% MAPs. The latter can be dissociated from tubulin by ion-exchange chromatography. The MAPs from brain can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 

Both dynein and MAP2 interact with microtubules at the same binding sites, namely, the C termini of a- and p-tubulin. Also, MAP2 inhibits the microtubule-activated ATPase of dynein and prevents microtubule gliding on dynein-coated glass coverslips. Thus, MAP2 and other fibrous MAPs may be regulators of microtubule-based motility in vivo (Paschal et al., 1989). 

The axoneme consists of a cylinder of nine outer doublets of fused microtubules and a pair of discrete central microtubules (commonly referred to as the 9 + 2 arrangement of microtubules). The outer doublets each consist of a complete A-microtubule and an incomplete B-microtubule, the deficiency in the wall of the latter being made up by a sharing of wall material with the former. The tip of the axoneme contains the plus ends of all of the constituent microtubules. Two curved sidearms, composed of the MAP protein dynein, are attached at regular intervals to the A-microtubules of each fused outer doublet (Figures 1 and 2). 

Paschal, B.M., Shpetner, H.S., Vallee, R.B. (1987). MAP IC is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J. Cell Biol. 105, 1273-1282. 

Gotoh, Y., Nishida, E Matsuda, S., Shiina, N., Kosako, H Shiokawa, K., Akiyama, T., Ohta, K., and Sakai, H. (1991 a). In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature 349 251-254. 

FIGURE 8.11 Multiple signal-transduction pathways initiated by calmodulin. Calmodulin bound to Ca2+ interacts and activates many enzymes, opening up a wide range of possible cellular responses. Abbreviations MAP-2, microtubule-associated protein 2 NO, nitric oxide Tau, tubulin assembly unit. 

The very beginning of the first mitotic cell cycle of the mouse embryo seems to be controlled by the mechanisms characteristic for both meiotic and mitotic cell cycles. Active MAP kinase, its substrate p90rsk and the CSF activity itself could influence the cellular processes within the one-cell embryo. Indeed, we have observed that despite the entry into the interphase (as judged by the low activity of MPF) some proteins are actively phosphorylated as during the meiotic M phase (e.g. 35 kDa complex Howlett et al 1986, Szollosi et al 1993), the nuclei and the microtubule interphase network start to form only 1.5 hours after activation (Szollosi et al 1993). This delay in the phenomena characteristic for the interphase could be linked to the mixed meiotic/mitotic character of this early period. This delay probably allows the correct transformation of the sperm nucleus into the male pronucleus. In species like Xenopus or Drosophila the transitional period between the meiotic and the mitotic cell cycle control is probably much shorter since it is proportional to duration of the short first cell cycle of these rapidly cleaving embryos. Mammalian embryos are perhaps the most suitable to study this transition because of the exceptionally long first embryonic cell cycle. 

Kalab P, Kubiak JZ, Verlhac MH, Colledge WH, Maro B 1996 Activation of p90Kk during meiotic maturation and first mitosis in mouse oocytes and eggs MAP kinase-independent and dependent activation. Development 122 1957—1964 Kubiak JZ, Weber M, dePennart H, Winston N, Maro B 1993 The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF. EMBO J 12 3773-3778... 

Verlhac MH, de Pennart H, Maro B, Cobb MH, Clarke HJ 1993 MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev Biol 158 330-340 Verlhac MH, Kubiak JZ, Clarke HJ, Maro B 1994 Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes. Development 120 1017-1025... 

In favorable systems, the coherent movement of neuro-filaments and microtubule proteins provides strong evidence for the structural hypothesis. Striking evidence was provided by pulse-labeling experiments in which NF proteins moved over periods of weeks as a bell-shaped wave with little or no trailing of NF protein. Similarly, coordinated transport of tubulin and MAPs makes sense only if MTs are being moved, since MAPs do not interact with unpolymerized tubulin [31]. 

Inactivation of the tau gene by homologous recombination results in mice that are largely normal [22], indicating that tau is a nonessential protein. This may reflect functional redundancy. Thus, mice doubly deficient in tau and the microtubule-associated protein MAP IB exhibit nervous system defects that are more severe than those in the MAP IB single knockout line. 

Filamentous tau is hyperphosphorylated. This is an early event that appears to precede filament assembly. It also renders tau unable to interact with microtubules. Much effort has gone into the mapping of phosphorylation sites and the identification of candidate protein kinases and phosphatases. For sites that are also phos-phorylated in normal brain tau, a higher proportion of tau molecules is phosphorylated in filamentous tau. In addition, filamentous tau is phosphorylated at more serine and threonine residues than tau from normal adult brain. Phosphorylation-dependent anti-tau antibodies were instrumental for the study of many phosphorylation sites. In particular, phosphorylation of S214 and S422 was found to be specific for assembled tau. 

MAP microtubule-associated protein NGFR nerve growth factor receptor... 

The reaction of anti-neurofilament antibodies with high molecular weight aggregates from rat neuronal cytoskeletal proteins provided direct evidence for neurofilament cross-linking after 2,5-hexanedione administration (Lapadula et al. 1986). Immunoblotting with antibodies specific for phosphorylated forms of cytoskeletal proteins has demonstrated a reduction of phosphorylation in neurofilament proteins and microtubule-associated-protein 2 (MAP-2) after 2,5-hexanedione treatment (Abou-Donia et al. 1988). 

In resting neutrophils it is estimated that there are about 11-23 microtubules per cell, with a diameter of approximately 25 nm and a wall width of 5 nm. They are long, tubular structures made by the helical formation of tubulin molecules, which are either a- or /3-subunits, each with a relative molecular mass of 55 kDa (Fig. 4.9). Each subunit is present in equimolar amounts in a tubulin molecule, and these subunits exist as dimers of one a- and one /3-subunit. Because microtubules are polar, growth of the fibre is biased towards one end, termed the plus end. A number of microtubule-associated proteins (MAPs) affect the dynamic shape of the microtubule, and in the resting neutrophil about 35-40% of the tubulin pool is assembled, whilst the remainder can be assembled very rapidly after cell stimulation. 

The stuctural integrity of the neuron is maintained by a protein cytoskeieton. Microtubules are the thickest cytoskeietai eiements and have an anionic poiarity due to their subunit arrangement. They are composed of microtubuie-associated proteins (MAPs), and provide support to the axons and dendrites. Neurofilaments are the most abundant cytoskeietai eiements, and are composed of proteins caiied cytokeratins. In the pathoiogy of Aizheimer s disease, neurofiiaments are known for their characteristic tangied appearance. Microfilaments are the thinnest cytoskeietai eiements and are composed of actin. 

There has been an intensive examination of the biochemical properties of microtubule proteins over the past 15 years, and most of the work has focused on proteins derived from neural systems. For convenience, we will deal with the molecular properties of tubulin first and then collectively consider the so-called microtubule-associated proteins (MAPs). 

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