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C A Collins

Publications and source records attributed to C A Collins.

At least 37 records · Page 2Linked to original sources
PubMed1992

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.

Adenosine Triphosphatases↗
PubMed1990

Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins.

A complementary DNA encoding the D100 polypeptide of rat brain dynamin--a force-producing, microtubule-activated nucleotide triphosphatase--has been cloned and sequenced. The predicted amino acid sequence includes a guanine nucleotide-binding domain that is homologous with those of a family of antiviral factors, inducible by interferon and known as Mx proteins, and with the product of the essential yeast vacuolar protein sorting gene VPS1. These relationships imply the existence of a new family of GTPases with physiological roles that may include microtubule-based motility and protein sorting.

Amino Acid Sequence↗
PubMed1989

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.

Adenosine Triphosphatases↗
PubMed1987

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.

Alkaloids↗
PubMed1986

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.

Adenosine Triphosphatases↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Adenosine Triphosphatases↗
PubMed1985

Use of multiple monoclonal antibodies to characterize the major microtubule-associated protein in sea urchin eggs.

Microtubules assembled from sea urchin eggs with the use of taxol contain a 77,000-dalton protein as the major nontubulin component [Vallee and Bloom (1983): Proc Natl. Acad. Sci. U.S.A. 80:6259-6263]. We have raised five monoclonal antibodies to this protein to aid in its characterization. Immunoblot analysis of the sea urchin microtubule purification fractions indicated that the protein copurified quantitatively with microtubules. All five antibodies stained the mitotic spindle of dividing sea urchin eggs by immunofluorescence microscopy, indicating that the protein was a component of the mitotic spindle and suggesting that it was actually localized on microtubules in vivo. Immunofluorescent staining of higher resolution was observed in a subpopulation of the coelomic cells found in adult sea urchins, confirming that the 77,000-dalton protein is indeed present on microtubules in vivo. Because taxol was not used for the immunofluorescence experiments, we conclude that the microtubule-associated protein (MAP)-like behavior of the 77,000-dalton protein in vitro was not induced artifactually by taxol. To determine whether this protein is a component of sea urchin microtubules in general, cilia obtained from blastula stage embryos and sperm tail flagella were analyzed with the antibodies. The protein was undetectable by both immunoblot analysis and immunofluorescence microscopy in both preparations of axonemal microtubules. These results indicated that the 77,000-dalton MAP is restricted to cytoplasmic and mitotic microtubules in the sea urchin. Furthermore, in view of its particular abundance in embryos, whose microtubules are devoted substantially to mitosis, the 77,000-dalton MAP is likely to play an important role in regulating the activity of mitotic spindle microtubules in the sea urchin.

Alkaloids↗
PubMed1984

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.

Carotenoids↗
PubMed1984

Rapid diglyceride phosphorylation in isolated bovine rod outer segments.

When isolated bovine rod outer segment fragments were incubated with [gamma-32P]ATP, 32P, as revealed by autoradiography, was rapidly incorporated into rhodopsin bands on sodium dodecyl sulfate polyacrylamide gels, and into a low Mr lipid band. Incorporation of 32P into rhodopsin was light-dependent, but labeling of the lipid band was not. A single phosphorylated product, phosphatidic acid, was identified by 2-dimensional thin layer chromatography and by high pressure liquid chromatography of the corresponding glycerophosphate ester. Incorporation of label into phosphatidic acid was detected as early as 15 sec following start of incubation and the product was stable for at least 30 min. No other products were detected, indicating that under the experimental conditions phosphatidic acid was not metabolized to other phospholipids. Up to 1 mol phosphatidic acid was formed per 18 to 40 mol rhodopsin present.

Adenosine Triphosphate↗
PubMed1983

Identification of phosphatidylinositol kinase in rat liver lysosomal membranes.

Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate 32P from [gamma-32P]ATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phosphate based on its chromatographic behavior on Silica Gel H thin layer plates as compared with standard phosphoinositides. The deacylation product, glyceryl-phosphorylinositol phosphate, was compared with standards in chromatographic and electrophoretic systems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4,5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was active at neutral pH in the presence of 20 mM MgCl2. The addition of 0.4% Triton X-100 stimulated the enzyme activity toward endogenous substrate, and the highest activity was observed in the presence of detergent and 1 mM phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X-100. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergent-stimulated activity of liver microsomes and plasma membrane, the previously recognized sources of this enzyme in the liver cell.

1-Phosphatidylinositol 4-Kinase↗
PubMed1982

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes.

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.

Animals↗
PubMed1982

Temperature dependence of the sodium channel gating kinetics in the node of Ranvier.

Temperature dependence of the Na+ channel gating kinetics was measured from the ionic and charge displacement currents in the node of Ranvier of Xenopus laevis, m3h kinetics was applied, assuming a delay, delta t, in the activation process. The rate constants for the m- and h-process showed Arrhenius temperature-dependence with Q10 of 2.34 and 2.9 respectively, while delta t exhibited non-Arrhenius temperature-dependence. Q10 for PNa, measured as 1.6, was smaller than for the rate constants and similar to that for a diffusion process. A negative shift and decrease in voltage sensitivity of the steady-state curves, h infinity and m infinity, occurred with decreasing temperature. The maximum time constant obtained from a single exponential fit to the displacement currents during the pulse for times greater than 90 microseconds exhibited Q10 of 2.01, which lies between that for PNa and that for tau m.

Animals↗
PubMed1982

Activation and inactivation characteristics of the sodium permeability in muscle fibres from Rana temporaria.

1. The steady-state and kinetic characteristics of the processes of activation and inactivation of the Na(+) permeability, P(Na), were measured in cut skeletal muscle fibres from Rana temporaria under voltage-clamp conditions.2. The specific resistance, r(ss), in series with the surface sarcolemma, was estimated as 6 Omega cm(2) by measuring the initial value of the membrane potential transient in response to current pulses under current-clamp conditions. To reduce the error in the potential across the sarcolemma introduced by r(ss), Na(+) currents were recorded using positive feed-back compensation, in the presence of tetrodotoxin (2.4-5 nm).3. P(Na)(t) was fitted with m(3)h kinetics assuming a voltage-dependent delay, deltat, to the start of the activation process.4. The P(Na)-V(p) curve exhibited saturation at potentials more positive than 30 mV. m(infinity), calculated as (P(Na), (infinity)/ P(Na))((1/3)) as a function of V(p), was a sigmoid curve with a mid point at -35 mV. The slope, dm(infinity)/dV(p), at this point was 0.032 mV(-1).5. Using a double-pulse protocol a non-exponential time course for the development of fast inactivation at small depolarizations was observed.6. The time constant for activation, tau(m), as a function of V(p), and tau(h) as a function of V(p), could be fitted with an approximately bell-shaped function, maximum of 430 mus at -43 mV and 925 mus at -78 mV respectively, at 15 degrees C.7. The mid-point potential of the h(infinity)-V(l) curve occurred at -58 mV, and h(infinity) approached 1 for V(1) values more negative than -103 mV.8. Using a double-pulse procedure the development of a slow inactivation of the Na(+) current was demonstrated. Its time course could be described in terms of a single exponential function, time constant equal to 0.58 s. The recovery from slow inactivation could be described by a similar exponential for recovery times smaller than 1 s.

Animals↗
PubMed1982

Fast charge movements in skeletal muscle fibres from Rana temporaria.

1. Fast charge movements were measured in cut skeletal muscle fibres from Rana temporaria.2. The initial time course of the current in response to a sudden displacement of the membrane potential from -110 to -60 mV was analysed in terms of an electrical equivalent circuit modified from Falk & Fatt (1964).3. The specific resistance in series with the sarcolemma was estimated as 7.4 Omega cm(2). The total capacity (surface sarcolemma plus tubular membrane) was estimated as 3.43 muF/cm(2).4. The asymmetry currents settling within 1 ms during depolarizing pulses of increasing size (on-response), from a holding potential around -120 mV, could be described in terms of a single exponential. The asymmetry currents after the pulses (off-response) exhibited at least two components.5. The integral of the on-response, Q(on), as a function of V(p), could be fitted using a function of the Boltzmann type. At the mid-point of the distribution curve, equal to -38 mV, the slope was 0.012 mV(-1). A saturating value of 28 pC was reached at 40 mV.6. The off-response to pulses not exceeding 3 ms exhibited two components. The first one had an exponential time course. The charge Q(off) associated with this fast component was always equal to Q(on).7. tau(on) (the relaxation time constant), as a function of membrane potential was asymmetrical, exhibiting a maximum value of 233 mus at about -38 mV.8. For V(p) values smaller than -20 mV the Q(on)-V(p) and tau(on)-V(p) curves could be analysed using the two-state transition model. From this analysis the average transition potential V' was estimated as -38 mV and the effective valence of the mobile charges as 1.36.9. Double-pulse protocols (duration of pre-pulses referred to as T in the range 0-3 s) showed that Q(on) and tau(on) decreased as T increased. Single transient analysis shows that the changes are confined to the transient for depolarizing pulses.10. This immobilization of the charges is reversible and follows a similar time course to the slow inactivation of the Na(+) conductance described in the preceding paper.11. A differential effect of the depolarizing pre-pulse on the ionic and asymmetry currents is seen in the decrease of tau(on) with increasing T while tau(m) remains constant.

Animals↗