Lamin Drosophila

To purify lamins. Drosophila extracts prepared as described above should be supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lamins from these extracts should be batch adsorbed to antilamin/protein A/sepharose by incubation overnight at 4°C. The column should then be washed extensively with equilibration buffer and purified lamins recovered from the antilamin affinity column with elution buffer. After elution, the solution containing the lamins should be immediately neutralized with Na2HP04 added to a final concentration of 50 mM. Immunoaffinity-purified lamins should be aliquoted, frozen by immersion in liquid nitrogen, and stored at -70°C until use. 

Stuurman N, Sasse B, Fisher PA. 1996. Intermediate filament protein polymerization Molecular analysis of Drosophila nuclear lamin head-to-tail binding. J Struct Biol 117 1-15. 

Goldberg, M., Lu, H., Stuurman, N, Ashery-Padan, R., Weiss, AM., Yu, J., Bhattacharyya, D., Fisher, P.A., Graenbaum, Y. and Wolfner, M.F. (1998) Interactions among Drosophila nuclear envelope proteins lamin, otefin, andYA. Mol. Cell Biol. 18, 4315-4323. 

Drosophila genome none are in the yeast genome. Because the lamin, but not the cytosolic, groups of IPs are expressed in Drosophila, lamins are probably the evolutionary precursor of the IF superfamily. 

Drosophila polypeptides have been definitively and specifically identified in subnuclear fractions enriched for all karyoskeletal elements they have been specifically localized to these elements in situ. Hence, the Drosophila nuclear lamina contains lamin Dmo derivatives (Smith et al., 1987 McConnell et al., 1987 Gruenbaum et al., 1988), lamin C (Bossie and Sanders, 1993 Riemer et al., 1995), and otefin (Hard et al., 1989). Nuclear pore complexes contain gp210o (McConnell etal, 1987 Berrios etai, 1995) as well as polypeptides with immunochemical homology to myosin (Berrios and Fisher, 1986 Berrios et al., 1991 Berrios, 1992). The interphase chromosome scaffold contains large amounts of the enzyme, DNA topoisomerase II (Berrios et al, 1985 McConnell et al, 1987 Meller et al, 1995). 

Baricbeva, E. A., Berrios, M., Bogachev, S. S., Borisevich, I. V., Lapik, E. R., Sharakbov, I. V., Stuurman, N., and Fisher, P. A. (1996). DNA from Drosophila melanogaster j3-heterochFomatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ. Gene 171, 171-176. 

Bossie, C. A., and Sanders, M. M. (1993). A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein. J. Cell Sci. 104,1263-1272. 

Gruenbaum, Y., Landesman, Y., Drees, B., Bare, J. W., Saumweber H., Paddy, M. R., Sedat, J., Smith, D. E., Benton, B. M., and Fisher, P. A. (1988). Drosophila nuclear lamin precursor Dmo is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene. J. Cell Biol. 106, S77-S86. 

Lin, L., and Fisher, P. A. (1990). Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms. J. Biol. Chem. 265, 12596-12601. 

Rzepecki, R., Bogachev, S. S., Kokoza, E., and Sturrman, N. (1997). In vivo association of lamins with nucleic acids in Drosophila melanogaster. Submitted for publication. 

Figures 6 and 7 (see color plate for Fig. 7) show two uses we have made of the autocorrelation function to quantitate features of images. The micrographs in Fig. 6 show grazing optical sections of nuclei from a Drosophila early embryo and a HeLa cell stained with an antinuclear lamin antibody. Using an autocorrelation analysis of images like this, we demonstrated that the mean spacing between adjacent local maxima in the antilamin staining pattern was —0.5 ptm in four...

Fig. 11 Quantitative measures of lamin-chromatin separation distances in Drosophila early embryo nuclei, (a) Probability of lamin-chromatin separation distance as a function of difference in circumferential solid angle along the nuclear periphery (AS) and difference in radial position (Ar) (see Paddy et al., 1990, for more details). (A0, Ar) = (0, 0) represents the center of the nearest local maximum in the lamin staining pattern. The distance above the A0, Ar plane is proportional to the probability that one of the 93 peripheral chromatin loci is located A0, Ar from the nearest local maximum in the antilamin staining pattern. The maximum value in the plot represents a probability of 4%. (b) Probability of lamin-chromatin separation distance as a function of difference in circumferential solid angle (A0). This plot results from integrating the data in panel a along the radial (Ar) axis, (c) probability of lamin-chromatin separation distance as a function of total separation distance. This plot results from using the Ad, Ar) coordinates of each point in panel a to calculate the separation distance at which the observed probabilities occur. [Reprinted with permission from Paddy el al. (1990). Copyright Cell Press.]...

V. Cell-Free Disassembly of Drosophila Lamin Aggregates (Polymers)... 

Immunoaffinity purification of Drosophila lamins Dmi, Dm2, and Dm ,i, from either embryos or tissue culture cells can be performed essentially as described... 

Stage 14 Drosophila egg chambers (hereafter referred to as oocytes) are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei (purified from either embryos or tissue culture cells) as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins (Maus et al., 1995). Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. Cell-free nuclear lamina disassembly is ATP dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. 

Biochemical analyses can be performed in parallel with or instead of morphological studies. At various time points during the course of in vitro disassembly, aliquots of the reaction mixture should be separated into supernatant and pellet fractions by sedimentation at 12,000 g and proteins subjected to SDS-PAGE and immunoblot analysis. An extract of stage 14 Drosophila oocytes contains only lamin Dm i, (Smith and Fisher, 1989 see also Lin and Fisher, 1990 Mans et at., 1995). Purified Drosophila nuclei contain lamins Dmi and Dm2 (Smith et al, 1987 see also Lin and Fisher, 1990 Maus et al, 1995). Lamin Dmmit migrates with a gel mobility intermediate to lamins Dm, and Dm2 (Fig. 4). 

Fig. 4 Immunoblot analysis demonstrating mobility differences among different Drosophila lamin isoforms. Lane 1, the 20-cm long 7% polyacrylamide gel was loaded with stage 14 oocyte extract containing only lamin Dm ,i, lane 2, loaded with embryo nuclei containing both lamins Dmi and Dm2. The migration positions of the different lamin isoforms are indicated to the right of the figure.

Stuurman, N., Maus, N and Fisher, P. A. (1995). Interphase phosphorylation of the Drosophila nuclear lamin Site-mapping using a monoclonal antibody. J. Cell Sci. 108, 3137-3144. 

Some degree of controversy exists as to whether nuclear envelope formation precedes, parallels, or follows the assembly of a nuclear lamina (see Georgatos et ai, 1994 Lourim and Krohne, 1994). Formation of the nuclear envelope in Drosophila embryo extracts is lamin dependent (Ulitzur et ai, 1992). In contrast, lamin-independent nuclear envelope assembly in vitro has been reported in Xenopus (Newport et ai, 1990 Meier et ai, 1991) and sea urchin (Collas et ai, 1995). The latter studies corroborate immunofluorescence observations of nuclear reconstitution after mitosis in somatic mammalian cells in vivo (Chaudhary and Courvalin, 1993). Assembly of a nuclear lamina in vitro can be monitored by immunofluorescence and immunoblotting using anti-lamin antibodies. 

Ulitzur. N., Harel, A., Feinstein, N., and Gruenbaum, Y. (1992). Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract. J. Cell Biol. 119,17-25. 

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