Unidirectional microtubule assembly in cell-free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell-free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)- and slow (minus)- growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post-fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.     

Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995- 8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.     

Structural polarity and directional growth of microtubules of Chlamydomonas flagella

A basic question concerning microtubule assembly is the polarity of growth, namely, whether subunits can add to either end of a growing microtubule or whether growth proceeds by subunit addition to only one end. To approach this question in an in vitro system, experiments were carried out on the addition of microtubule subunits to isolated flagellar axonemes. Flagella were detached from Chlamydomonas by brief treatment with non-ionic detergent, isolated by differential centrifugation, and incubated with crude high-speed extracts of porcine brain tissue or with purified tubulin (obtained by repetitive temperature-dependent assembly and disassembly). Electron microscopy of negatively stained samples showed as many as 11 long microtubules added at one end of more than 90% of the axonemes. Colchicine (100 μm), CaCl₂ (2.5 mm), and low temperature (0 °C) both prevented and reversed microtubule assembly but had no effect on axonemal length. In crude extracts microtubules formed on both members of the axonemal central pair but on only the A-tubule of the outer doublets. Flagellar fragments, produced by mechanical shearing, were also incubated with microtubule subunit. Single tubules formed at only one end of outer doublet fragments; the appearance of single tubules on one or both members of central pair fragments was predominantly unidirectional. Structural analysis of frayed axonemes and the asymmetry of side-arm attachments permitted the absolute polarity of the axonemal fragments to be determined and revealed that assembly proceeded by addition of subunits to the distal ends of the axonemal microtubules. Using purified brain tubulin, a limited extent of proximal addition and growth on the B-tubule also occurred. The extent of proximal addition increased with increasing protein concentration and temperature. We conclude that the microtubules of flagella have an intrinsic polarity reflected in their side-arm attachments and in their directionality of growth.     

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.     

Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.     

Active motor proteins can couple cargo to the ends of growing microtubules

In living cells, dynamic microtubule ends interact with specialized protein complexes located on microtubule targets such as chromosomes and the cell cortex. A significant role in coupling microtubule ends to these complexes has been attributed to motor proteins, which are thought to provide a physical link while at the same time allowing for microtubule growth or shrinkage. In the past, motor-coated beads have been shown to be able to follow the ends of depolymerizing microtubules, in a direction opposite to their natural walking direction. Here we show that beads coated with plus-end-directed motors can also stay attached for several seconds to the ends of growing microtubules. Upon arrival at the microtubule end, fast-moving beads reduce their velocity to the microtubule growth velocity. We show that the tendency to stay attached depends on the initial bead velocity and that the microtubule growth velocity is unaffected by the presence of the bead.     

On and Around Microtubules: An Overview

Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.     

Membrane/microtubule tip attachment complexes (TACs) allow the assembly dynamics of plus ends to push and pull membranes into tubulovesicular networks in interphase Xenopus egg extracts

We discovered by using high resolution video microscopy, that membranes become attached selectively to the growing plus ends of microtubules by membrane/microtubule tip attachment complexes (TACs) in interphase- arrested, undiluted, Xenopus egg extracts. Persistent plus end growth of stationary microtubules pushed the membranes into thin tubules and dragged them through the cytoplasm at the approximately 20 microns/min velocity typical of free plus ends. Membrane tubules also remained attached to plus ends when they switched to the shortening phase of dynamic instability at velocities typical of free ends, 50-60 microns/min. Over time, the membrane tubules contacted and fused with one another along their lengths, forming a polygonal network much like the distribution of ER in cells. Several components of the membrane networks formed by TACs were identified as ER by immunofluorescent staining using antibodies to ER-resident proteins. TAC motility was not inhibited by known inhibitors of microtubule motor activity, including 5 mM AMP-PNP, 250 microM orthovanadate, and ATP depletion. These results show that membrane/microtubule TACs enable polymerizing ends to push and depolymerizing ends to pull membranes into thin tubular extensions and networks at fast velocities.     

Density and distribution of α-bungarotoxin-binding sites in postsynaptic structures of regenerated rat skeletal muscle

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold- treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold- treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.     

The polarity and stability of microtubule capture by the kinetochore

We have studied the capture of microtubules by isolated metaphase chromosomes, using microtubules stabilized with taxol and marked with biotin tubulin to distinguish their plus and minus ends. The capture reaction is reversible at both the plus and minus ends. The on rate of capture is the same for both polarities but the dissociation rate from the kinetochore is seven times slower with microtubules captured at their plus ends than those captured at their minus ends. At steady state this disparity in off rates leads to the gradual replacement of microtubules captured at their minus ends with those captured at their plus ends. These results suggest that the kinetochore makes a lateral attachment near the end of the microtubule in the initial capture reaction and shows a structural specificity that may be important in proper bipolar attachment of the chromosome to the spindle.     

Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.     

Distinct roles of doublecortin modulating the microtubule cytoskeleton

Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors.     

Asymmetric behavior of severed microtubule ends after ultraviolet- microbeam irradiation of individual microtubules in vitro

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.     

Direct observation of steady-state microtubule dynamics

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Inhibition of neurite initiation and growth by taxol

We cultured sensory neurons from chick embryos in media containing the alkaloid taxol at concentrations from 7 X 10(-9) to 3.5 X 10(-6) M. When plated at taxol concentrations above 7 X 10(-8) M for 24 h, neurons have short broad extensions that do not elongate on the culture substratum. When actively growing neurites are exposed to these levels of taxol, neurite growth stops immediately and does not recommence. The broad processes of neurons cultured 24 h with taxol contain densely packed arrays of microtubules that loop back at the ends of the process. Neurofilaments are segregated from microtubules into bundles and tangled masses in these taxol-treated neurons. At the ends of neurites treated for 5 min with taxol, microtubules also turn and loop back abnormally toward the perikaryon. In the presence of 7 X 10(-9) M taxol neurites do grow, although they are broader and less branched than normally. The neurites of these cells appear to have normal structure except for a large number of microtubules. Taxol probably stimulates microtubule polymerization in these cultured neurons. At high levels of the drug, this action inhibits neurite initiation and outgrowth by removing free tubulin from the cytoplasm and destroying the normal control of microtubule assembly in growing neurites. The rapid inhibition suggests that microtubule assembly may occur at neurite tips. At lower concentrations, taxol may slightly enhance the mechanisms of microtubule assembly in neurons, and this alteration of normal processes changes the morphogenetic properties of the growing neurites.     

The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore.     

XMAP215 is a processive microtubule polymerase

Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.