Microtubules (MTs), rigid and hollow cylindrical structures of about 25 nm diameter, are composed of a- and b-tubulin dimers. They determine cell shape and play important roles in diverse processes such as cell division, cell motility and migration, cellular transport, and signal transduction. Both a and b tubulins exist in several isotypic forms and can undergo several post-translational modifications. In higher eukaryotes at least 14 tubulin isotypes have been reported that are expressed in a tissue specific manner. MTs are polar structures with two distinct ends, a fast growing "plus" end and a slow growing "minus" end. In most cells, MTs are organized into a single array with their minus ends associated with a MT organizing center located near the nucleus, and their plus ends located toward the cell’s periphery near the plasma membrane. This gives the cell a defined polarity based on the inherent polarity of MTs. This polarity is utilized by the MT-associated motor proteins that move "cargo" to the minus or plus ends of cellular MTs. Tubulin dimers constantly polymerize and depolymerize, and MTs can undergo rapid cycles of assembly and disassembly. The first stage of MT formation, the nucleation phase, is slow. In the presence of Mg2+ and GTP, a and b tubulins join together in an end-to-end manner to form protofilaments with alternating a and b subunits. The second phase, also known as the elongation phase, proceeds rather rapidly. For tubulin heterodimerization and association of tubulins to form MTs, GTP must be bound to both a and b subunits. GTP bound to b-tubulin is hydrolyzed to GDP during or immediately after polymerization. This weakens the binding affinity of tubulin for adjacent molecules and favors depolymerization that contributes to the dynamic behavior of MTs. Heterodimers can add or dissociate at either end of a MT; however, there is greater tendency for addition to occur at the faster growing plus end where b-tubulin is exposed. MTs also undergo “treadmilling,” in which tubulin molecules bound to GDP are continually lost from the minus end and are replaced by the addition of GTP-bound tubulin molecules to the plus end of the same MT. During the formation of MTs, the alternating elongation and shortening cycles provide dynamic instability that is critical for directing MTs towards target sites, such as kinetochores, focal adhesions, and migrating membranes. Dynamic instability, a tightly regulated phenomenon, is particularly critical for the remodeling of the cytoskeleton during mitosis. It is characterized by four important variables: the rate of MT growth, the rate of shortening, the frequency of transition from the growth state to shortening, and the frequency of transition from shortening to growth. The growth and shortening of a MT is dependent upon the rate of tubulin addition relative to the rate of GTP hydrolysis. Tubulin-bound GTP is hydrolyzed to tubulin-GDP + Pi and tubulin-GTP is added to the plus end almost simultaneously. However, when GTP-bound tubulin molecules are added more rapidly than GTP is hydrolyzed, the MT retains a GTP cap at its plus end and the growth continues. When the rate of polymerization declines, the GTP bound to tubulin at the plus end is hydrolyzed to GDP and the GDP-bound tubulin dissociates, resulting in rapid depolymerization and shrinkage of MT.  |
The inherent dynamic instability of MTs can be modified by the interactions with MT-associated proteins (MAPs) and MT- regulatory proteins. For example, MAPs can bind to MTs and increase their stability, while other proteins act to disassemble MTs, by increasing the rate of tubulin depolymerization. The best-characterized MAPs are MAP-1, MAP-2, and tau proteins. The activity of MAPs is tightly regulated by their phosphorylation state and altered phosphorylation state of MAPs has been positively linked to the pathogenesis of Alzheimer’s disease. Growth factor signals can activate protein kinases that catalyze phosphorylation of tubulin-binding domains of MAPs and allow them to detach from MTs. XMAP215, a highly conserved MAP of 215 kDa, plays an important role in controlling MT dynamics during cell cycle. It stabilizes the plus ends of MTs, promoting growth at the plus end and preventing catastrophic shrinkage. At the onset of mitosis, higher phosphorylation of XMAP215 results in increased MT instability causing them to disassemble. During the end of mitosis, protein phosphatase activity predominates as the MT array of interphase is re-established. Given their essential role in the formation of the mitotic spindle during cell division, MTs have been very attractive targets for cancer chemotherapy. Anti-mitotic agents that can selectively disrupt MT dynamics, either by targeting a specific tubulin isotype or a particular stage of cell division have great therapeutic value as chemotherapeutic agents. These agents exploit the difference in MT dynamics between rapidly dividing cancerous cells and normal cell populations. For example, drugs such as colchicine and colcemid bind tubulin and inhibit MT polymerization, thus blocking mitosis. On the other hand, agents such as taxol stabilize MTs and prevent cell division.
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