Temperature-dependent reversible assembly of taxol-treated microtubules.

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

Processing, characterisation and biocompatibility of iron-phosphate glass fibres for tissue engineering.

Iron-phosphate glass fibres based on the CaO-Na2O-Fe2O3-P2O5 system have been processed and characterised via thermal, XRPD, dissolution rates, diameter and biocompatibility studies. The compositions investigated were fixed at 50mol% P2O5, and the CaO content was varied between 30, 35 and 40mol%. The Fe2O3 was added in low amounts from 1-5mol%, substituting it for the Na2O mol%. The number of Tc (crystallisation temperature) peaks detected from the thermal analysis traces only showed correlation with XRPD analysis, for five out of the 15 compositions investigated. It has been suggested that either the crystalline phases had very similar Tc temperatures or that the other phase(s) were present in very small quantities. There was a good match seen with number of Tm (melting temperature) peaks picked up from the DTA traces, with the number of phases identified from XRPD analysis. The main phases identified from XRPD were NaCa(PO3)3, CaP2O6 and NaFeP2O7. Using network connectivity (NC), predictions on Qn species present within the compositions investigated were made. The predicted species (metaphosphates) matched with phases identified from XRPD analysis. A decrease in dissolution rates for the bulk glass and glass fibres was seen with an increase in CaO mol%, along with an increase in Fe2O3 mol%. An increase in fibre dissolution rates was seen with a decrease in diameter size. The biocompatibility studies were conducted using a conditionally immortal muscle precursor cell line derived from the H-2Kb-tsA58 immortomouse. It was found that iron-phosphate glass fibres containing 4-5mol% Fe2O3 was sufficient for cell attachment and differentiation. It was seen that myotubes formed along the axis of the fibres (which was indicative of differentiation). The biocompatibility of these compositions was attributed to the enhanced chemical durability of the glass fibres.

Isolation of mitotic microtubule-associated proteins from sea urchin eggs.

We have used a taxol-based microtubule purification procedure and monoclonal antibodies to isolate and characterize the MAPs of mitotic spindle microtubules in the fertilized sea urchin egg. In so doing, we hope to have identified some of the essential working parts of the mitotic apparatus, namely those proteins that regulate the assembly, disassembly, organization and mechanochemical properties of spindle microtubules. The results of this effort strongly suggest that a rich diversity of polypeptides associate with mitotic spindle microtubules. Whether each of these represents an individual protein species is not currently known. It is possible, for example, that particular spindle MAPs comprise multiple, distinct subunits. This would not be surprising in light of the facts that both MAP-1 and MAP-2 contain lower molecular weight subunits, and that axonemal dyneins are complex assemblies of several polypeptide species. Our future efforts with the sea urchin system will be to determine how the various mitotic spindle MAPs we have identified function individually and in concert, and how those functions contribute to the mechanochemical properties of the spindle.

Emotionally expressive coping predicts psychological and physical adjustment to breast cancer.

This study tested the hypothesis that coping through emotional approach, which involves actively processing and expressing emotions, enhances adjustment and health status for breast cancer patients. Patients (n = 92) completed measures within 20 weeks following medical treatment and 3 months later. Women who, at study entry, coped through expressing emotions surrounding cancer had fewer medical appointments for cancer-related morbidities, enhanced physical health and vigor, and decreased distress during the next 3 months compared with those low in emotional expression, with age, other coping strategy scores, and initial levels on dependent variables (except medical visits) controlled statistically. Expressive coping also was related to improved quality of life for those who perceived their social contexts as highly receptive. Coping through emotional processing was related to one index of greater distress over time. Analyses including dispositional hope suggested that expressive coping may serve as a successful vehicle for goal pursuit.

Role of beta-catenin in epidermal stem cell expansion, lineage selection, and cancer.

The mammalian epidermis is an excellent model with which to analyze the factors that regulate adult stem cell renewal, lineage selection, and tumor formation. One of the key regulators of all three processes is beta-catenin, the main cytoplasmic effector of the canonical Wnt signaling pathway. In this chapter, we review some of the ways in which beta-catenin exerts its effects on cultured human epidermal cells and in genetically modified mice. We highlight the importance of the timing and level of activation and discuss some of the pathways activated downstream from beta-catenin. Finally, we demonstrate the importance of Lef/Tcf-independent beta-catenin signaling through interaction with the vitamin D receptor.

Characterization of the sea-urchin egg microtubule-activated ATPase.

We have found that cytoplasmic extracts from unfertilized sea-urchin eggs contain a prominent microtubule-activated ATPase activity. This activity is induced by polymeric tubulin, but not by tubulin subunits. The activity cosediments with taxol-stabilized microtubules in an ATP-independent manner. We have separated the ATPase from cytoplasmic dynein and other ATPases on sucrose gradients. The sedimentation, enzymic and microtubule-binding properties of the microtubule-activated species show it to be distinct from cytoplasmic dynein, myosin and kinesin. Since the major function of microtubules in the early sea-urchin embryo is in mitosis, this enzyme represents a new candidate for a role in spindle motility.

A microtubule-activated ATPase from sea urchin eggs, distinct from cytoplasmic dynein and kinesin.

We report an ATPase activity, present in sea urchin egg cytosol, that is activated by microtubules. The activity sediments at 10 S in sucrose gradients and is clearly distinct from activities at 12 S and 20 S due to cytoplasmic dynein. Potent activation of the ATPase is observed when endogenous egg tubulin is induced to assemble with taxol or when exogenous taxol-stabilized pure brain tubulin microtubules or flagellar outer-doublet microtubules are added. No activation by tubulin subunits or taxol alone is detectable. In contrast to flagellar or cytoplasmic dynein, the microtubule-activated enzyme is unaffected by vanadate or by nonionic detergents and hydrolyzes GTP in addition to ATP. In contrast to kinesin, it cosediments with microtubules in the presence or absence of ATP. The microtubule-activated enzyme may have a role in microtubule-based motility.

Preparation of microtubules from rat liver and testis: cytoplasmic dynein is a major microtubule associated protein.

A microtubule associated protein from brain tissue (MAP 1C), has been found to possess many properties in common with ciliary and flagellar dyneins (Paschal et al.:J. Cell Biol. 105:1273-1282, 1987). However, this protein, now designated as cytoplasmic dynein, exhibited several properties which distinguish it from axonemal forms of the enzyme. We have investigated these characteristics further in a study of cytoplasmic dyneins from non-neuronal tissues. Rat liver and testis in particular were found to contain high levels of cytoplasmic dynein. The yield of dynein from testis was over 70 micrograms/g of tissue, making this the best source of cytoplasmic dynein of all tissues so far examined. The characterization of dynein from these sources has confirmed and extended our previous observations concerning the unique properties of cytoplasmic dynein. Activation of liver and testis dynein occurred at low (less than 1 mg/ml) tubulin concentration. Polypeptides identified as subunits of brain cytoplasmic dynein (74, 59, 57, 55, and 53 kDa) were present in liver and testis preparations. In addition, polypeptides at 150 and 45 kDa were found to copurify with the non-neuronal dyneins. The liver and testis enzyme hydrolyzed pyrimidine nucleotides at rates up to 12.5 times faster than ATP, though the relative affinity of cytoplasmic dynein for CTP was much lower (Km = 1.0 mM) than that for ATP. The properties of the testis enzyme were consistent with its identification as a cytoplasmic dynein rather than a sperm axonemal precursor. These data indicate that cytoplasmic dyneins may be widespread in distribution and that they share certain biochemical properties unique from those of axonemal dyneins. These characteristics are consistent with the proposal that cytoplasmic dynein plays a universal role in retrograde organelle motility.

Determination of vitamin A and vitamin A acetate by high-performance liquid chromatography with fluorescence detection.

A rapid and sensitive high-performance liquid chromatographic method with fluorescence detection has been devised for the analysis of vitamin A (retinol) and its acetate. The method employs a C18 reversed-phase column and methanol as an eluent. The detection of these two compounds is monitored with fluorescence excitation at 348 nm and emission at 470 nm. Detector noise established the lower limit of quantitation at approximately 0.5 ng. Plasma samples were employed to evaluate the accuracy, reproducibility, and applicability of the method. Less than 1 ng of vitamin A in plasma (as low as 1 microliter) can be quantitated by this procedure.

Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells.

Polyclonal antisera have been raised against cytoplasmic dynein purified from calf brain and rat testis. These antibodies reacted most strongly with the 74 kDa dynein intermediate chain, but also recognized the 410 kDa heavy chain, and the 150 and 45 kDa polypeptides previously observed to copurify with cytoplasmic dynein from rat tissues. Localization studies were performed by indirect immunofluorescence microscopy using a fibroblast cell line. Dynein-specific staining appeared vesicular, distributed throughout the cell, but more concentrated near the nucleus. Double-labeling studies using fluorescent markers for membranous organelles indicated a co-localization of dynein with lysosomes. The distribution of the dynein-positive lysosomes was disrupted by treatment of the cells with microtubule-active drugs, and by acidification of the cytoplasm. Comparison of the distribution of lysosomes with peripheral microtubules indicated a high degree of coincidence. These results are consistent with the hypothesis that cytoplasmic dynein is involved in retrograde-directed movement of membranous organelles. In mitotic cells, dynein staining was also apparent along the microtubules of the mitotic apparatus, though vesicular staining was still conspicuous. The presence of dynein on vesicles as well as on spindle microtubules indicates that dynein distribution between these compartments may be regulated by distinct binding proteins.

The question remains: is the spliceosome a ribozyme?

The two phosphoryl transfer steps of pre-mRNA splicing are catalyzed within the large ribonuclear protein machine called the spliceosome. The highly dynamic nature of the spliceosome has presented many challenges to a structural and mechanistic understanding of its catalytic core. While much evidence supports the popular hypothesis that the catalytic steps of pre-mRNA splicing are mediated by spliceosomal RNA, a role for protein in catalysis cannot yet be ruled out. A highly conserved protein, Prp8, is a component of the catalytic core. We review data consistent with the hypothesis that Prp8 functions as a cofactor to an RNA enzyme.

Genetic interactions between the 5' and 3' splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing.

The YAG/ consensus sequence at the 3' end of introns (the slash indicates the location of the 3' splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5' end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5' /GUA with Y-3 of the 3' YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5' /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y-3 in the 3' YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y-3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5' /GUA and the GAG of U6 function in binding the 3' YAG/ during the second catalytic step.

Microtubule-independent phospholipid stimulation of cytoplasmic dynein ATPase activity.

In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.

Dynamin forms polymeric complexes in the presence of lipid vesicles. Characterization of chemically cross-linked dynamin molecules.

Dynamin is a GTP-binding protein that is involved in the release of coated endocytic vesicles from the plasma membrane. We have been characterizing the enzymatic properties of purified rat brain dynamin to better understand how GTP binding and hydrolysis relate to its proposed function. Previously, we have demonstrated that activation of dynamin GTPase results from positive cooperative associations between dynamin molecules as they are bound to a polymeric surface. Our present report has extended these studies and has examined the structural features of dynamin self-association. After treatment with the zero-length protein cross-linking reagent, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, dynamin in solution was found cross-linked into dimers. This homodimer likely reflects the native soluble state of the molecule. After binding to brain vesicles, dynamin was cross-linked into higher order oligomers of greater than 800 kDa. Dynamin, copurified on brain membranous organelles, also formed multimeric complexes when cross-linked suggesting dynamin exists in polymeric form in vivo. No cross-linked species other than homo-oligomers were observed, providing no evidence for close interactions between dynamin and membrane proteins. From experiments examining the effects of GTP, GDP, guanosine 5'-3-O-(thio)triphosphate, and 5'-guanylyl-beta,gamma-imidodiphosphate on cross-linking, we have determined that both dynamin membrane binding and self-association occur independently from the nucleotide-bound state of the enzyme. An 80-kDa dynamin fragment that is lacking its carboxyl-terminal domain is not cross-linked into higher order oligomers, suggesting that this domain is required for binding of dynamin to membranes and the subsequent enhancement of oligomerization. However, the dynamin fragment was found to form dimers indicating that this domain is not required for dynamin dimerization. Cross-linked dynamin was able to cooperatively bind microtubules, but did not exhibit GTPase activation. We propose that intramolecular cross-links in the dynamin monomer impart structural constraints that prevent the enhancement of GTP hydrolysis. We describe a model of the dynamin activation process to be considered in further investigations of the role for dynamin in endocytic vesicle formation.