Methods Actin

Simon, J.R., and Taylor, D.L. (1988) Preparation of a fluorescent analog Acetamidofluoresceinyl labeled dictyostelium discoideum a-actin. In Methods in Enzymology, (R.B. Vallee, ed.), Vol. 134, p. 47. Academic Press, San Diego. 

A basic structural property of protein filaments is polarity, that is, directionality. Almost all naturally occurring filaments are polar (e.g., F-actin, microtubules, TMV, and so on). The few exceptions are either bipolar, like myosin (Huxley, 1963 Squire, 1981), or nonpolar, like intermediate filaments (Herrmann and Aebi, 2004). One method of determining... 

Hizume K, Yoshimura SH, Maruyama H, Kim J, Wada H, Takeyasu K (2002) Chromatin reconstitution development of a salt-dialysis method monitored by nano-technology. Arch Histol Cytol 65 405 13 Hizume K, Yoshimura SH, Takeyasu K (2004) Atomic force microscopy demonstrates a critical role of DNA superhelicity in nucleosome dynamics. Cell Biochem Biophys 40 249—262 Hizume K, Yoshimura SH, Takeyasu K (2005) Linker histone HI per se can induce three-dimensional folding of chromatin fiber. Biochemistry 44 12978-12989 Hofmann WA, de Lanerolle P (2006) Nuclear actin to polymerize or not to polymerize. J Cell Biol 172 495-496... 

In this approach, bisulfite-treated DNA is used as the template, and 50-100nM nnmethylated or methylated DNA-specific primers are nsed for PCR amplification in separate reactions. For quantification, a AAC.J, method is nsed and normalized with the C.J, (cycle threshold) for the 3-actin gene (43). Alternatively, a fragment of the target gene promoter will be amplified using primers designed from a CpG-free area as an internal control (see Note 3). To eliminate any primer dimer that will compromise the accuracy of the results, an additional step in the PCR cycles above... 

Lawrence, J. B. and Singer, R. H. (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 13, 1777-1799. 

Method Signal a[polymer] Signal noise ratio Sensitivity to length Shear rate Native actin Expense Sample size (ml)... 

Figure 3. Critical concentration behavior of actin self-assembly. For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by methods that measure the sum of addition and release processes occurring at both ends. Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscosity measurements. Forthe bottom curves, the polymerization behavior is typically determined by fluorescence assays conducted under conditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1 -actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs). Note further that the barbed end (or (+)-end) has a lower critical concentration than the pointed end (or (-)-end). This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition to the (+)-end at the expense of the subunit loss from the (-)-end.

Other assays are being explored in HTS platforms to identify potential inhibitors of invasion, for example, compounds that affect the shape or morphology of cells or their ability to generate invadopodia. Quintavalle et al. recently described such a method initially using Src-transformed NIH 3T3 fibroblasts grown on 384-well optical plates. Imaging of cellular and nuclear morphology combined with phalloidin-stained F-actin was used to discriminate compounds which reduced (or enhanced) the number of... 

Recently, a method has been developed to microinject cell-polymer suspensions into 3-D collagen matrices in a 96-well plate format. This was used to perform a proof-of-principle screen of known signaling pathway inhibitors in murine breast cancer cells. The cells were visualized by staining of the actin cytoskeleton and effects of the compounds measured using multiparametric imaging. The system also proved amenable to the use of enzymatically... 

The luminous zone in deton appears to have a small finite thickness This was given by Mitchell Paterson (Ref 1) as being less than 0.24 cm for NG, with duration of deton flame of less than 0.3 microseconds. Herzberg Walker (Ref 2) have measured the duration of actinic radiation by highspeed camera methods and from these calculated that the luminous zones in the deton of HE s were from 0.03 to 0.09 cm thick (Ref 5, pp 154-55)... 

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