Cold-stable Microtubules Research Articles

Purification and assay of a 145-kDa protein (STOP145) with microtubule-stabilizing and motility behavior.

The capacity of microtubules to disassemble in vitro is profoundly affected by a protein factor designated STOP (stable tubule only polypeptide). Here we report the isolation of STOP protein and confirm that its activity is, as predicted, highly substoichiometric to the tubulin in microtubules. The isolation of the 145-kDa STOP (STOP145) protein has been effected from isolated cold-stable microtubules by two column steps: DEAE ion-exchange and a calmodulin affinity column. To confirm the protein's activity we have produced an antibody against STOP145 and have used the antibody to specifically remove the protein and the activity using an antibody-linked affinity column. We conclude that the STOP145 protein accounts for the observed in vitro stabilization of microtubules.

17] Purification and assay of cold-stable microtubules and STOP protein

17] Purification and assay of cold-stable microtubules and STOP protein

Identification of endogenous calmodulin-dependent kinase and calmodulin-binding proteins in cold-stable microtubule preparations from rat brain.

Calmodulin-dependent kinase activity was investigated in cold-stable microtubule fractions. Calmodulin-dependent kinase activity was enriched approximately 20-fold over cytosol in cold-stable microtubule preparations. Calmodulin-dependent kinase activity in cold-stable microtubule preparations phosphorylated microtubule-associated protein-2, alpha- and beta-tubulin, an 80,000-dalton doublet, and several minor phosphoproteins. The endogenous calmodulin-dependent kinase in cold-stable microtubule fractions was identical to a previously purified calmodulin-dependent kinase from rat brain by several criteria including (1) subunit molecular weights, (2) subunit isoelectric points, (3) calmodulin-binding properties, (4) subunit autophosphorylation, (5) calmodulin-binding subunit composition on high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (6) isolation of kinase on calmodulin affinity resin, (7) kinetic parameters, (8) phosphoamino acid phosphorylation sites on beta-tubulin, and (9) phosphopeptide mapping. Endogenous cold-stable calmodulin-dependent kinase activity was isolated from the microtubule fraction by calmodulin affinity resin column chromatography and specifically eluted with EGTA. This kinase fraction contained the calmodulin-binding, autophosphorylating rho and sigma subunits of the previously purified kinase. The rho and sigma subunits of this kinase represented the major calmodulin-binding proteins in the cold-stable microtubule fractions as assessed by denaturing and non-denaturing procedures. These results indicate that calmodulin-dependent kinase is a major calmodulin-binding enzyme system in cold-stable microtubule fractions and may play an important role in mediating some of the effects of calcium on microtubule and cytoskeletal dynamics.

Cold-stable microtubules and microtubule-organizing centres in astrocytes in primary culture

Immunocytochemical staining with anti-tubulin antibodies showed cold-stable microtubules in astrocytes in culture. On rewarming, microtubule-organizing centres (MTOC) could be visualized. MTOCs showed structural complexity, from 2–3 fibres to a mass of interweaving fibres. Only one MTOC per cell was found in cultures of both polygonal and stellate-bearing cells.

Spatial organization of axonal microtubules.

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.

Isolation from bovine brain of a superstable microtubule subpopulation with microtubule seeding activity.

Cold-stable microtubule protein isolated from beef brain is capable of seeding microtubule assembly under conditions that prevent the initiation of self-assembly of cold-labile microtubules. We have developed a quantitative assay for the determination of seeding activity. Using this assay, we find that seeding activity is apparently due to microtubule fragments that resist -80 degrees C, a condition that causes the depolymerization of cold-stable microtubules ("cold stability" is defined as resistance to 0 degree C disassembly), but rapidly depolymerize when exposed to 3.0 mM free calcium, to micromolar Ca2+-calmodulin, or to 0.2 M NaCl at 4 degrees C. After salt treatment, seeding activity is permanently lost although microtubule cold stability is retained through further assembly cycles. Similarly, after sedimentation of microtubule seeds the supernatant protein assembles into cold-stable microtubules, which are permanently devoid of seeding activity. By contrast, seeding activity can be recovered by recycling of supernatant protein from preparations exposed to 3.0 mM calcium or to Ca2+-calmodulin prior to centrifugation, indicating the solubilization of an active component (designated "preseeds") under these conditions. Polyacrylamide gels show some differences in polypeptides between seeding and non-seeding cold-stable microtubule preparations. Approximately 35% of the microtubule population assembled from beef brain crude extract is cold stable, while approximately 2% constitutes -80 degrees C resistant seeds. The formation of seeds from seed-forming subunits (preseeds) occurs rapidly, is apparently a cooperative phenomenon, and occurs on preexisting microtubules under either assembly initiating or steady-state conditions.

In Purkinje cell dendrites of the young rat, thyroid hormone controls the resistance of microtubules to fixation at low temperature

Microtubules were studied in parallel fibres and Purkinje cell dendrites of 14-day-old normal and propylthiouracil-treated rats after fixation of the cerebellum at room and low temperature (4°C). In both control and propylthiouracil-treated animals, nearly all the microtubules of parallel fibres disappeared after fixation at low temperature. In Purkinje cell dendrites of control animals, 60% of the microtubules persisted at low temperature. Propylthiouracil treatment led to a 78% decrease in the density of these cold-stable microtubules. In contrast, the density of cold-labile microtubules remained normal. Administration of low doses of thyroxine to the propylthiouracil-treated rats produced a rapid increase in the density of cold-labile microtubules, but long-term treatment with the hormone was necessary to obtain normal density of cold-stable microtubules. Thyroid hormone thus seems to regulate some properties of microtubules involved in their resistance to fixation at low temperature. These properties could be of importance in growth and branching of dendrites.

Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation.

Cold-labile microtubule protein can be rendered cold-stable by addition of a fraction containing a small number of polypeptides that are derived from cold-stable microtubules. These polypeptides can be obtained from purified cold-stable microtubules by passage through a DEAE-cellulose (DE-52) ion exchange column from which they emerge in the first eluate fraction. The stabilizing activity of these proteins is abolished by phosphorylation catalyzed by two types of protein kinases, one dependent on calmodulin and the other independent of that regulatory protein. The calmodulin-dependent reaction appears to phosphorylate mainly two polypeptides, 56 and 72 kilodaltons; the reaction is blocked by trifluoperazine. The calmodulin-independent reaction appears to phosphorylate different cold-stable microtubule-associated proteins. That reaction is observed only in purified material obtained from vigorously homogenized brain tissue. Gently homogenization yields cold-stable microtubules that are responsive only to the calmodulin-dependent protein kinase. A distinguishing feature of the calmodulin-independent reaction is that it does not occur on polypeptides while they are bound to the microtubules.

Purification and characterization of sheep brain cold-stable microtubules.

The isolation of cold-stable microtubules in high yields, described previously only from rodents, was extended to the brain of higher animals. Under optimal conditions, yields of 30 mg of cold-stable microtubles per 100 g of sheep brain could be obtained routinely. Material purified by two polymerization cycles displayed the same stability to cold temperature or to millimolar concentrations of calcium and the same lability to calmodulin and to ATP as did the purified material obtained from the rat [Job, D., Rauch, C.T., Fischer, E.H. & Margolis, R.L. (1982) Biochemistry 21, 509]. Furthermore, DE-52 chromatography of this material yielded a fraction that restored cold stability when added to cold-labile microtubules. Known to bind to calmodulin and to enhance microtubule assembly, tau proteins had no cold-stabilizing activity. Protein profiles of the cold-stabilizing fraction from sheep and rat brain were similar to one another but showed no protein bands corresponding to the tau proteins.

Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks.

A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.

The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells.

When metaphase PtK1 cells are cooled to 6-8 degrees C for 4-6 h the free, polar, and astral spindle microtubules (MTs) disassemble while the MTs of each kinetochore fiber cluster together and persist as bundles of cold-stable MTs. These cold-stable kinetochore fibers are similar to untreated kinetochore fibers in both their length (i.e., 5-6 micrometer) and in the number of kinetochore-associated MTs (i.e., 20-45) of which they are comprised. Quantitative information concerning the lengths of MTs within these fibers was obtained by tracking individual MTs between serial transverse sections. Approximately 1/2 of the kinetochore MTs in each fiber were found to run uninterrupted into the polar region of the spindle. It can be inferred from this and other data that a substantial number of MTs run uninterrupted between the kinetochore and the polar region in untreated metaphase PtK1 cells.

Rapid disassembly of cold-stable microtubules by calmodulin.

Purified cold-stable microtubules from the rat brain are insensitive to podophyllotoxin and to millimolar concentrations of free calcium. However, in the presence of calmodulin at concentrations substoichiometric to that of tubulin, calcium causes rapid microtubule disassembly. The half-maximal effective calcium concentration in the presence of calmodulin is 100 microM. With 800 microM free calcium, the half-maximal effective concentration of calmodulin is 1.0 microM (or one-tenth the tubulin concentration). Calmodulin is without effect in the absence of calcium. Troponin C is approximately one-fifth as effective as calmodulin, and parvalbumin is totally ineffective. Troponin I partially inhibits the calcium/calmodulin-induced disassembly of microtubules in the crude extract and blocks the calcium/calmodulin effect on purified cold-stable microtubules. A 5-fold excess of trifluoperazine does not inhibit the calcium/calmodulin-induced disassembly.

Characterization of rat brain crude extract microtubule assembly: correlation of cold stability with the phosphorylation state of a microtubule-associated 64K protein.

We have conducted preliminary investigations into the control of microtubule assembly in rat brain crude extract supernatants. The rationale for these experiments is that microtubules interact with many proteins and are undoubtedly subject to physiological control mechanisms that are lost during tubulin purification. A more complete understanding of the cellular regulation of microtubules must include the physiology of these proteins. Assembly can be monitored in rat brain crude extract high-speed supernatants by measuring the increase in solution turbidity. We find that assembly is maximal in both rate and extent in the absence of added nucleotide. Increasing concentrations of either adenosine 5'-triphosphate (ATP) or guanosine 5'-triphosphate (GTP) inhibit both initiation and elongation of microtubules. GTP appears necessary for assembly and is apparently replenished from an intrinsic energy source during the time course of the assembly reaction. Inhibition of GTP production prevents microtubule assembly, and addition of exogenous GTP will reverse the blockage. Enzymatic removal of GTP at steady state causes a rapid depolymerization to the cold-stable microtubule level. Both GTP production and microtubule assembly display periodic oscillatory maxima. Cold-stable microtubules, which are always present in rat brain crude extract preparations, are rapidly made labile by addition of ATP. Analysis of proteins in cold-stable and cold-labile microtubule fractions shows changes in protein phosphorylation but not in the microtubule-associated protein composition. The tentative conclusion is that the state of phosphorylation of a 64K protein, designated the "switch protein", determines the cold stability or lability, and therefore the dimer association and dissociation rates, of crude extract microtubules.

Cold stable microtubules in brain studied in fractions and slices.

Microtubules were shown to remain intact in brain slices and subfractions maintained at 0 degrees C for 1 h. Under the same conditions, microtubules isolated from brain by warm assembly-cold disassembly methods, disassemble into their constituent subunit proteins. No selective depletions of microtubules were seen when brain slices were incubated in homogenizing buffer at either 0 degrees C or 37 degrees C. The response of native microtubules in brain slices in incubation in other solutions showed that their properties were otherwise the same as those of assembled microtubules. The separated alpha and beta subunits of isolated cold labile and cold stable microtubules were compared by electrophoresis and isoelectric focusing and were shown to possess the same mobilities. The results suggest that native microtubules are temperature insensitive and that isolated microtubules are assembled from pre-existing pools of subunit proteins. The results further suggest that native microtubules possess a factor, lacking in isolated assembled microtubules, which confers temperature stability on the former.

Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase.

In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concurrently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region. To test the dynamic equilibrium model of chromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber microtubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated. Abrupt changes in temperature from 22 degrees C to as low as 4 degrees C or as high as 31 degrees C in fact produced immediate changes in chromosome velocity to new constant velocities. No transient changes in velocity were observed. At 4 degrees C (10 degrees C for grasshopper cells), chromosome movement ceased. Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little. The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between temperature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber microtubules. On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways of polymerization and depolymerization.

Cold-stable microtubules in the cytoplasm of mouse embryo fibroblasts

Treatment of cultured mouse embryo fibroblasts with Triton X-100 after prolonged incubation at 0 °C reveals a network of microtubules in the cytoplasm of cooled cells. This network of cold-stable microtubules was demonstrated by immunofluorescence microscopy, using a monospecific antibody against tubulin and by electron microscopy. The cold-stable microtubules, as well as the ordinary cytoplasmic microtubules, were sensitive to Ca ions and were not observed in the cells pre-treated with colchicine or vinblastine. The cold-stable microtubules do not seem to be in equilibrium with the pool of depolymerized tubulin at 0 °C.

Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells.

Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells.