Nucleated Polymerization Research Articles

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative.

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.

Microtubule treadmills and their possible cellular functions.

The polymerization of microtubules in vitro exhibits many of the properties of a nucleated helical polymerization reaction of the type described by Oosawa and Asakura (1975) for the polymerization of actin. Assembly requires a nucleation event (about which little is known) that results in the formation of a short microtubule fragment. Growth and shortening of microtubules occur by addition and loss of tubulin subunits at the ends of the microtubules. Early studies by Allen and Borisy (1974) and Binder et al. (1975) demonstrated that growth at the two microtubule ends in vitro occurs at substantially different rates, thereby establishing that microtubules possess an intrinsic growth polarity.

Calorimetric studies of the in vitro polymerization of brain tubulin.

The enthalpy change for chain propagation in the polymerization of bovine tubulin has been studied directly by stopped-flow microcalorimetry at 17 degrees and 25 degrees, and found to be 0 +/- 1 kcal per mol of 6S tubulin dimer at both temperatures. Substantial heat evolution with a half-time of decay of approximately 1 hr was observed w-en tubulin was injected into the calorimeter. This heat was shown to result from contamination of the tublin by small amounts of some material from the crude brain homogenate from which the tubulin was prepared, and to be totally unconnected with microtubule assembly. Model calculations of nucleated polymerization processes reveal that the van't Hoff enthalpy calculated from the temperature dependence of the critical polymerization concentration is a complicated function of the separate enthalpy changes for nucleation and chain propagation. The published values of this quantity for tubulin probably pertain primarily to the nucleation process. It is shown that our observation of a propagation enthalpy change of vanishingly small size is not necessarily inconsistent with the reported van't Hoff enthalpies.

Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly.

The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoué could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.

2846. Haloether formation from formaldehyde not a working problem: Kallos, G. J. & Solomon, R. A. (1973). Investigations of the formation of bis-chloromethyl ether in simulated hydrogen chloride-formaldehyde atmospheric environments. Am. ind. Hyg. Ass. J.34, 469

Aβ accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aβ nucleated polymerization is essential for Aβ fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aβ seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aβ seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aβ-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aβ42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aβ seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

Heterogeneous Nucleation of Polymer Melts on High-Energy Surfaces. II. Effect of Substrate on Morphology and Wettability

Heterogeneous Nucleation of Polymer Melts on High-Energy Surfaces. II. Effect of Substrate on Morphology and Wettability

Heterogeneous nucleation of polymer melts on surfaces. I. Influence of substrates on wettability

Heterogeneous nucleation of polymer melts on surfaces. I. Influence of substrates on wettability