Although molecular components of signal transduction pathways are rapidly being identified, how elements of these pathways are positioned spatially and how signals traverse the intracellular environment from the cell surface to the nucleus or to other cytoplasmic targets are not well understood. The discovery of signaling molecules that interact with microtubules (MTs), as well as the multiple effects on signaling pathways of drugs that destabilize or hyperstabilize MTs, indicate that MTs are likely to be critical to the spatial organization of signal transduction. Microtubule organizing centres (MTOCs), which include fungal spindle pole bodies and centrosome in higher eukaryotes, are a structurally diverse group of organelles that share a conserved role in microtubule nucleation and spindle formation. However, recent studies propose that the function of MTOC components extends far beyond these established roles. Numerous cell cycle regulators, checkpoint proteins and microtubule plus tip binding proteins localize to MTOCs during the cell cycle, suggesting that these organelles serve as cellular scaffolds. In addition, several MTOC components such as γ -tubulin and its associating proteins have been directly implicated in the control of cell cycle progression, activation of checkpoint responses and the regulation of microtubule organization and dynamics.
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Introduction Microtubules are essential cytoskeletal polymers that are made of repeating α/β-tubulin heterodimers and are present in all eukaryotes. Microtubules affect cell shape, cell transport, cell motility, and cell division. All of these functions involve the interaction of microtubules with a large number of microtubule-associated proteins (MAPs), which are important for the regulation and distribution of microtubules in the cell. Of special interest are motor proteins of the kinesin and dynein families, which use ATP hydrolysis to move cargoes along microtubules or microtubules with respect to each other. Each microtubule is formed by the parallel association of protofilaments, linear polymers of tubulin dimers that are bound head to tail. The tubulin sequence and structure contain the information required for the self-assembly of protofilaments into polar, dynamic microtubules, which in turn interact with a variety of cellular factors (20). Microtubules are highly dynamic and can switch stochastically between growing and shrinking phases, both in vivo and in vitro. This nonequilibrium behavior, known as dynamic instability (20), is based on the binding and hydrolysis of GTP by tubulin subunits. Each tubulin monomer binds one molecule of GTP. The binding to α-tubulin at the N site is nonexchangeable, whereas the binding to β-tubulin at the E site is exchangeable. Only dimers with GTP in their E site can polymerize, but, after polymerization, this nucleotide is hydrolyzed and becomes nonexchangeable.
Although the complexity of microtubule regulatory pathways is not understood properly, the use of fluorescent derivatives
and characterization of taxol-resistant tubulin mutants, with our growing knowledge of the structure of the tubulin-taxol complex, have improved much about molecular details. Tubulin Dimer Structure The structure of the tubulin dimer at 3.7-Å resolution (Figure 1a) has been obtained by electron crystallography of zinc-induced tubulin sheets stabilized with taxol (20).The N-terminal nucleotide-binding domain (residues 1-206) is formed by the alternation of parallel beta strands (S1-S6) and helices (H1-H6). The nucleotide-binding pocket is formed by each of the loops connecting each strand and helix (loops T1-T6) and the N-terminal end of the core helix (H7). After the core helix is a smaller, second domain that is formed by three helices (H8-H10) and a mixed beta sheet (S7-S10). The C-terminal region is formed by two antiparallel helices (H11 and H12) that cross over the previous two domains. In the dimer the nucleotide in the α-subunit (GTP) is buried at the intradimer interface, readily explaining the nonexchangeability of the site (Figure 2b). The nucleotide at the E site is partially exposed on the surface of the dimer, allowing its exchange in solution. Figure 1 (a) Ribbon diagram of the electron crystallography structure of the tubulin dimer. Nucleotides are shown in pink and taxol in yellow. MAPs Microtubule-associated proteins (MAPs) are proteins that interact with the microtubules of the cellular cytoskeleton. MAPs bind to the tubulin subunits that make up microtubules to regulate their stability. A large variety of MAPs have been identified in many different cell types, and they have been found to carry out a wide range of functions. These include both stabilizing and destabilizing microtubules, guiding microtubules towards specific cellular locations, cross-linking microtubules and mediating the interactions of microtubules with other proteins in the cell. Some of them restricted to particular cell types. For example isoforms of MAP1 and MAP2 are expressed primarily in neurons, and MAP7 is restricted to epithelial cells (14).Aberrant expression of MAPs and their relevance to the resistant phenotype of a wide range of malignancies to microtubule-targeting agents have been documented (14). MTs interact with a number of binding proteins and regulation can occur at many levels. For more details about this you can refer the article S. Honore. et al (2005). (b) Structural elements surrounding the N-site nucleotide at an intradimer interface. Loops T1-T6 correspond to α-tubulin; loop T7* corresponds to the β-subunit within the dimer.
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Is MAPs having any role in cell cycle? Gene ontology study of MAPs from Drosophila embryos, shown that 9% proteins involving in cell cycle/mitosis (11). Remaining MAPs also have variety of functions. By using RNAi studies also it's demonstrated that many MAPs significantly affects the cell cycle (). Mainly five genes (CG7033, CG8231, CG8258, CG8351, and CG5525) showed monopolar spindles with reduced MT density in addition to generally low numbers of MTs in interphase cells, in relation to wild-type cells.
Figure 2 Functional Classification of 270 Drosophila Embryonic MAPs
Microtubules and signal transduction In the past decade, studies of the cytoskeleton have fused, almost by stealth, with studies of signal transduction. It has become clear that the cell's system of cytoskeletal filaments and its network of signaling pathways are intimately linked and function cooperatively to generate a cell phenotype tailored to the immediate conditions of the cell. In addition, when cells are remodeled the cytoskeleton is probably both effect and cause: it responds to signals; it organizes signaling pathways in space; and it may perform signaling functions itself. Because it comprises linear elements that span the cell, the cytoskeleton is well-constructed for integrating information.
We will see one elegant example that how microtubule responds to the extracellular signal. Fibroblasts in cell culture provide a relatively simple system for studying how extracellular signals reorganize and polarize the microtubule cytoskeleton. Grown in the absence of serum, fibroblasts contain mainly labile microtubules which grow and shrink rapidly. But when serum is restored to the medium, the cells produce an array of stable microtubules (6), which can be detected by their post-translational modifications and resistance to depolymerizing drugs. These microtubules extend from the microtubule organizing center (MTOC) near the nucleus to the periphery of the cell, and are oriented specifically toward the leading edge of cells at a wound site in the cell monolayer. The MTOC itself also moves to a position between the nucleus and the leading edge of the cells (1). Mechanisms for microtubule signal transduction
MT sequestering and release, MT delivery and MT scaffolding of signaling molecules (figure 3). The binding of CI-Costal 2 and
Figure 3. Three basic mechanisms for MT-mediated signal transduction are shown. The signaling factor, represented by a sphere, (a) is shown to interact directly with the MT , (b) via a motor protein or (c) via a putative scaffolding factor. (a) For sequestering and release, three possible release mechanisms are envisioned: (i) signal induced modification (represented by a ) of the factor itself, (ii) of the MT or (iii), enhanced MT depolymerization. (c) The scaffolding factor (represented by a cylinder) is shown to undergo a conformation change upon binding to the MT and this generates a binding site for the signaling factor. Other mechanisms (e.g. post-translational modification) may activate the binding of the scaffolding and signaling factors. NF B-I B to the MT surface may be examples of the sequestering and release mechanism (). Activation and release of MT bound signalling factors could be accomplished by: modification of the factor, modification of the MT and/or breakdown of the MT. The latter two release mechanism are specific are specific for MTs and there is evidence that MT dynamics might be regulated by signal transduction. Each of these mechanisms might also work in the reverse direction, to sequester inhibitory factors during signal transduction.
MT-mediated delivery could act either by delivering signaling factors to other components on the MT surface or to specific sites in the cell. MLK2-KIF3 may be an example of the former, and an unidentified motor in the Wnt signaling pathway may be an example of the latter. Activation of this mechanism by signal transduction could occur through enhanced motor-cargo interaction, enhanced motor-MT interaction, or stimulation of the motor itself. Kinesin polypeptides are known to be phosphorylated and this may regulate their interaction with cargo (12, 13). Post-translational modification of tubulin enhances kinesin binding (5, 10), and may contribute to the specific interaction of kinesin with stabilized MTs.
Drugs that breakdown MTs (e.g. colchicine, nocodazole, vinblastine) and drugs that hyperstabilize MTs (e.g. taxol and taxotere) have specific effects on diverse cellular processes involving signal transduction, for example, cell proliferation, gene expression, receptor signaling, apoptosis, and cell polarization (Table 1) (7). These drugs generally bind specifically to tubulin and have relatively few other direct cellular targets, implying that MTs may be involved
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Table 1. Effects of agents that break down or stabilize microtubules on signal transduction regulated process
in at least some of the signaling pathways regulating these processes. For example, MT depolymerizing agents stimulate proliferation, whereas MT stabilizing agents inhibit proliferation, suggesting some sort of MT-mediated sequestering and release mechanism. MTOC and γ- tubulin Microtubule organizing center (MTOC) is a general term describing a class of specialized structures that direct the assembly, orientation and organization of microtubules in eukaryotic cells. The role of MTOC as a central organizer of the microtubule cytoskeleton has been known for over a century, yet the mechanism by which MTOCs accomplished this role remained a mystery for some time. In recent years, a wealth of information has emerged. This is largely due to the discovery of γ- tubulin, a new member of the tubulin super-family (15). Numerous studies indicate that MTOCs and in particular, γ- tubulin containing complexes, function in additional microtubule processes other than regulating microtubule assembly and structure. A number of proteins involved in cell cycle regulation, checkpoints and microtubule organization and function to localize to MTOCs and appear to be regulated at these sites in a manner that is important for their function (fig 4). For example, mutations affecting the γTuSc appear to have consequences on microtubule organization and dynamics (4, 24, 25). MTOCs and cell cycle progression
During the cell cycle, the precise timing of key cellular processes such as spindle placement, mitosis and cytokinesis is essential for high fidelity chromosome segregation. Temporal organization of these events is coordinated by a group of proteins collectively termed cell cycle regulators. Figure 4 γ tubulin and MTOC components function in various cell cycle processes In recent years, studies in yeast have revealed that many regulators localize to the spindle pole bodies (SPBs) during the cell cycle (Fig. 4). As a result, the involvement of SPB components in the coordination of cell cycle events have been investigated and suggest additional roles for SPB components in spindle positioning, mitotic exit and cytokinesis. Spindle positioning. SPBs may influence the timing and execution of cellular processes by acting as scaffolds that promote interactions between regulatory proteins and their substrates at a critical place and time during the cell cycle. One example of this coordination is pre-anaphase spindle placement in the budding yeast (Fig. 5). The microtubule organizing protein Kar9 is asymmetrically recruited to the bud-bound SPB (SPBb) where it facilitates spindle alignment by guiding astral microtubules emanating from this pole into the bud (16-18). During this time, it is imperative that Kar9 remain asymmetric as the loss of Kar9 asymmetry targets both SPBs towards the bud.
Figure 5. Emerging roles of SPB components. (A) Numerous cell cycle regulators, checkpoint proteins and proteins involved in spindle placement localize to the SPBs. Some of these proteins such as Kar9 asymmetrically localize to only one SPB while others such as Bim1 symmetrically localize to both SPBs.
(B) SPBs are important for proper spindle positioning as they harbor many components of the Kar9 spindle positioning machinery. Intrinsic asymmetries between the two SPBs may contribute to the asymmetric localization of Kar9 by inhibiting Kar9 localization at the SPBm and promoting its localization to the SPBb. One possibility is that SPBs facilitate an interaction between Kar9 and the Cdc28/cyclin complex which regulates phosphorylation of Kar9 and its asymmetric localization. Regulation and formation of Kar9 complexes at SPBs is important for proper interactions between microtubule plus ends and the bud cortex. (C) Spindle placement is monitored by the spindle positioning checkpoint (SPC) which consists of a number of proteins that localize to SPBs. Events at the SPBm prolongs the localization of the Bub2/Bfa1 at both SPBs which inhibits Tem1 from promoting mitotic exit. What restricts Kar9 to one SPB is poorly understood, yet several lines of evidence suggest that SPBs may be the scaffolding sites for the regulatory machinery that governs Kar9 asymmetry. One factor known to contribute to the establishment of Kar9 asymmetry is phosphorylation of Kar9, mediated by the cell cycle regulatory kinase Cdc28, its associated early B-type mitotic cyclins(Clb3, Clb4 and Clb5), and the microtubule associating protein Bik1 (Fig. 5) (16-18).
Several mechanisms have been proposed to explain how Kar9 phosphorylation at the SPB translates into its asymmetric distribu-tion. One hypothesis is that phosphorylation of Kar9 at Ser197 and Ser496 by Cdc28/Clb4 occurs specifically at the mother-bound SPB (SPBm), which decreases its affinity for Bim1 at this pole; thereby promoting the relative enrichment of Kar9 at the SPBb. Alternate studies propose that phosphorylation of Kar9 occurs at the SPBb via Cdc28/Clb5 and Bik1, and enhances Kar9's specific accumulation at the bud-bound pole and its association with microtubule +ends. Though the precise players and effects of phosphorylation in these mechanisms differ, both proposed mechanisms highlight a common intriguing theme; that inherent asymmetries of the SPBs may influence the ability of Cdc28 and the cyclins to regulate Kar9 (Fig. 5). While the mechanism that drives asymmetry of the SPB remains controversial, the results of these studies are consistent with previous findings that have identified inherent functional and biochemical polarities between the two SPBs with respect to their association with additional proteins, their nucleating capabilities and their cellular inheritance following cytokinesis.
Similarly, asymmetric distribution of cytoplasmic dynein, which is required for maintaining the position of the spindle
during anaphase, and for pre-anaphase spindle placement in the absence of Kar9, is also a necessary requirement for proper spindle orientation in yeast. Control of dynein asymmetry to the SPBb is important for the placement of this pole into the bud during anaphase elongation and is partially dictated by the SPB component Cnm67 and Cdc28 in combination with the late Btype cyclins, Clb1 and Clb2. In addition, the respective roles of early and late B-type cyclins in Kar9 and dynein function ensures that both mechanisms for positioning the spindle are not active at the same time. Thus, the SPBs can be viewed as serving a central role in coordinating early and late spindle placement by scaffolding Cdc28 with different cyclins.
Mitotic exit and cytokinesis. Proper placement of the spindle and the SPBb is required for the onset of anaphase and exit from mitosis in budding yeast. In the budding yeast, mitotic exit occurs via a protein signaling cascade called the Mitotic Exit Network (MEN). The MEN pathway triggers the release of the evolu-tionarily conserved phosphatase Cdc14 from the nucleolus, which de-phosphorylates and regulates MEN components and other targets that regulate the metaphase-anaphase transition and cytokinesis (2, 9, 23). Many MEN components localize to one or both of the SPBs during mitotic exit, suggesting that SPBs scaffold this regulatory machinery (Fig. 5). Moreover, the SPB localization of these proteins was found to have functional relevance on mitotic exit. For example in budding yeast, entry of the SPBb into the bud results in an accumulation of the MEN components Tem1 and Cdc15 to the SPBb and loss of Bub2, all of which coincide with mitotic exit. Importantly,localization of Tem1 and Cdc15 to the SPBb is facilitated by the SPB components Nud1 and Cnm67 (3, 8).This demonstrates that for budding yeast, the establishment of mitotic exit depends on a spatial and temporal coordination between SPB placement and localization of the MEN machinery. In fission yeast, a pathway parallel to the MEN known as the Septation Initiation Network or SIN, similarly involves the local-ization of SIN components to SPBs, however the role of the SIN primarily facilitates the proper timing of cytokinesis. During division, contraction of the cytokinetic actomyosin ring (CAR) mediates septation and is coordinated with spindle formation and chromo-some segregation (19). SPBs scaffold the polo-like kinase Plo1 and the SIN component Cdc7, both of which are required for proper CAR formation and septation. Loss of this localization results in a cell cycle delay due to activation of the spindle assembly checkpoint. Additionally, SPBs also scaffold the SIN component Cdc11 which is required for the subsequent localization and activation of other downstream SIN components. Collectively, studies in budding and fission yeasts identify the SPBs as evolutionarily conserved scaffolding sites for proteins involved in spindle placement, mitotic exit and cytokinesis. The precise temporal and spatial localization of these regulatory pathways to SPBs suggests that as scaffolding sites, SPBs promote or maintain local environments that facilitate the ability of these proteins to govern cell cycle progression. Intriguingly, the involvement of SPBs in cell cycle progression may be more complicated than just a scaffolding role.
Spindle positioning checkpoint (SPC). In budding yeast, SPBs have been implicated in the checkpoint that monitors spindle placement. This checkpoint, termed the spindle positioning checkpoint (SPC), utilizes the small G-protein Tem1 as a master switch for either delaying or promoting mitotic progression. When spindle placement is perturbed, Tem1 is maintained in an inactive state, thereby
inhibiting the MEN until proper spindle placement is achieved. Many studies have shown Tem1 inactivation to be partly maintained through the GAP activity of the GTPase activating heterodimer Bub2/Bfa1 (Fig. 5).Recently, SPBs have also been implicated in mediating Tem1 inactivation, as Tem1 inhibition was dependent on the persistent localization of Bub2/Bfa1 to the SPBm rather than on their GAP activity. SPBs are proposed to be regulatory scaf-folding sites for events that mediate Tem1 activation. As of yet, the mechanism between Tem1 activation and the persistent localization of Bub2/Bfa1 to the SPBm remains unknown. However, interactions between Bub2/Bfa1 and Nud1 may provide the link in under-standing the participation of SPBs in this checkpoint. A role for SPBs in the SPC is further demonstrated by their ability to scaffold the checkpoint kinase Kin4. In response to spindle positioning defects, Kin4 inhibits the MEN by promoting the activity of Bub2/Bfa1 (). This is accomplished by counteracting the activity of the polo-like kinase Cdc5, which activates the MEN through inhibitory phosphorylation of Bfa1. In response to spindle misalignment, Kin4 and Bub2/Bfa1 complex are localized together at both poles and Kin4 is able to maintain Bub2/Bfa1's activity. Localization of Kin4 to SPBs also requires the SPB component Nud1, thus directly implicating a SPB component as a scaffold for members in this particular checkpoint. Conclusion
New evidence suggests that MT-associated proteins may also have roles in signal transduction. MTs themselves respond to signal transduction and this may be an important aspect of the integration and polarization of signaling pathways. Many other cellular processes are affected by drugs that interfere with MT dynamics and stability and one of the future challenges will be to identify the molecular basis for these effects. The role of MTOC components throughout the life cycle of the cell is an exciting and complex area of study. The attachment of both spindle and cytoplasmic microtubules to these sites supports an ability of these organelles to act as master controls for cell cycle progression, eliciting a checkpoint response, and coordinating microtubule organization during these events. The localization of cell cycle regulators, checkpoint proteins and microtubule plus tip binding proteins suggests that MTOCs have a scaffolding function whereby they spatially coordinate protein interactions which translate into downstream events at the cortex and/or microtubule end. More interesting is the possibility that MTOC components directly participate in a more active role in these processes such as participating in protein signaling cascades. Studies are revealing that components of γ- tubulin complexes appear to be prime candidates for mediating the role of MTOCs in these various cell cycle events. References
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