Cytoplasmic extract

Belmont LD, Hyman AA, Sawin KE, Mitchison TJ (1990) Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62 579-589... 

V/hen concentrated solutions of cytoplasmic extracts of certain cells are warmed to room temperature, the proteins form gels... 

Fig. 2 a) Results of gel fraction assay on cytoplasmic extracts of Ehrlich ascites cells (after Ishiura and 0kada(13)). 

Elasticity measurements already have been used to detect the presence of crosslinking proteins in cytoplasmic extracts(24). If preformed actin chains are employed, assessment of the rheological state of a network directly provides information about the parameters of crosslinking. Moreover, chain growth can bg studied, although indirectly, through the effects of Cq, Uq, on the concentration of crosslinking sites Sy. 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

Figure 12.5 Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and permeabilized with digitonin as described in Wilson et al., 1999. Fluorescein-PNA-labeled plasmids (containing the SV40 enhancer, 4.2 kb) or rhodamine-labeled BSA-NLS peptide conjugates were incubated with the cells for four hours at which time they were viewed by fluorescence microscopy. With no additions, neither DNA nor protein was imported, but in the presence of nuclear and cytoplasmic extracts both substrates localized to the nuclei. While plasmids containing the SV40 enhancer were taken up by the nuclei, those lacking the sequence were excluded. The remaining panels demonstrate the need for both the import machinery (importins and Ran) and a source of adapter proteins (nuclear extract) for plasmid nuclear entry, but not for protein nuclear localization.

Although researchers realized in the early 1950s that protein biosynthesis can continue after cell disruption, the first cell-free translation systems were made from the cytoplasmic extract of rat liver cells (11). Later, cell-free translation systems from E. coli cells were prepared (12,13). However, these systems were based on the endogenous mRNA remaining attached to ribosomes while the extract was prepared. The landmark in the history of cell-free translation was the translation of an external source of mRNA in E. coli extracts (14). The authors demonstrated that the pre-incubation of extracts at physiological temperature could eliminate endogenous mRNA from the extract ribosomes in the extract were found to accept either exogenous natural mRNA or synthetic polyribo-... 

Fig. 9. HiTrap chelating column used to purify histidine tagged glutathione-S-transferase from cytoplasmic extract.

This procedure describes the preparation of nuclear and cytoplasmic extracts from cells grown in suspension (2-201) and is essentially as described by Dignam et al.1 and modified by Jamison and Garcia-Bianco.2 A procedure for small-scale preparation of extracts from HeLa cells grown as monolayer has been described by Lee and Green.3 The latter procedure is recommended when the cell material is scarce, if radioactively labelled extracts are made, if several extracts from parallel cultures, or if expensive growth conditions are necessary for cell propagation. 

The IRE-binding activity was first monitored in rat cytoplasmic extracts by an electrophoretic mobility shift assay [40]. Soon thereafter, this activity was detected in most tissues and cell lines examined, and reported in the literature with various confusing names IRE-binding protein (IRE-BP) [79], iron regulatory factor ... 

Cytoplasmic extract Nuclear extract Normal human sera Calf sera... 

This chapter describes and discusses methods used in our laboratory to investigate each step of male pronuclear formation in a sea urchin egg cytoplasmic extract. When available, procedures previously reported for surf clam male pro-nuclear assembly in vitro are also indicated. These protocols may be applicable to other marine invertebrates as well. 

Cytoplasmic extracts have been made from immature oocytes, either unactivated or artificially activated (Longo et aL, 1994). Ovaries are dissected from ripe females and incubated in seawater to release germinal vesicle (GV)-stage oocytes. Oocytes are washed three times in seawater by suspension and spinning in a hand centrifuge. 

A cytoplasmic extract consists of a crude egg lysate containing cytosol, membrane vesicles (MVs), ribosomes, and occasional mitochondria, but is free of pronuclei, essentially all yolk granules, and other large cytoplasmic inclusions. Unfertilized egg extracts support only partial chromatin decondensation (Cameron and Poccia, 1994), whereas extracts from fertilized eggs support full chromatin decondensation and nuclear envelope assembly (Zhang and Ruderman, 1993 Cameron and Poccia, 1994 Collas and Poccia, 1995a,b). 

Surf Clam. Lamin assembly in clam male pronuclei occurs almost invariably in extracts made from 65-min-activated eggs (Longo et ai, 1994). For chromatin decondensation and nuclear envelope assembly, no exogenous nucleotides are required. These are presumably supplied by the cytoplasmic extract. Approximately half of the decondensed sperm nuclei in unactivated or 15-min-activated extracts assemble variable deposits of lamin. 

Preparation of Cytoplasmic Extracts from Mammalian Cells... 

We have found that cytoplasmic extracts from a number of cell types support nuclear import in vitro. The extracts are prepared essentially as described in Dignam et al. (1983) using cells grown on a large scale in spinner cultures or purchased from a commercial source as a frozen cell pellet. We routinely start with approximately 10 cells. The nuclei produced in the initial steps of the procedure can be used as a source of snRNPs. 

An in-vitro nuclear system prepared from HeLa cells, described by Friedman and Mueller (1968), appears to continue the DNA replication process observed in vivo. The system requires intact nuclei, the four deoxyribonucleoside triphosphates, magnesium ion, ATP, and, in addition, a heat-labile cytoplasmic factor. The activity of the system was similar to the DNA synthetic activity observed in intact cells in synchronized culture (Friedman and Mueller, 1968). Cytoplasmic factors also appear to stimulate in-vitro nuclear systems prepared from normal and regenerating rat livers (De Beilis, 1969). The cytoplasmic factors are present in both normal and regenerating liver cytoplasm and stimulate nuclear DNA synthesis in both systems. The stimulation was most marked using regenerating liver factors and normal liver nuclei (De Beilis, 1969). When mouse liver nuclei are recombined with cell free cytoplasmic extracts from mouse ascitic or L-cells active in DNA synthesis there is a marked stimulation of DNA synthesis in the isolated nuclei (Thompson and McCarthy, 1968). Cytoplasmic preparations from HeLa cells also stimulated DNA synthesis in mouse liver nuclei. 

Fig. 12. Informosomes from loach embryo extracts labeled for 3 hours with uridine. Dotted lines, radioactivity solid lines, optical density. A. Sucrose density gradient centrifugation of cytoplasmic extract (22,000 rpm for 18 hours). B. Recentrifugation of the material of 73 to 62S peak in CsCI density gradient. C. The same but of 45 to 36S zone. (From Ovchinnikov et al. 1960a. Molec. Biot. (U.S.S.RJ, 3 448 3.)...

Fig. 13. Nucleoprotein complexes containing virus-specific RNA in the cytoplasm of HeLa cells infected with vaccina virus. A. Sucrose density gradient centrifugation of cytoplasmic extract, after 30-minute labeling with uridine. Centrifugation 18.5 hours at 22,000 rpm. B. Recentrifugation of RNP isolated from SOS zone sucrose gradient in CsCI density gradient. Centrifugation in preformed CsCI density gradient in SW-39 rotor at 37,000 rpm, 20 hours. (From Belitsina et al. 1968. Molec. BioL (U.S.S.R.), 2 727-735.)...

The presence of informosomelike RNPs has been demonstrated also in the case of Ehrlich ascites carcinoma cells infected with Sendai virus (Volkova et al., 1969). The cytoplasmic extracts of the cells labeled for 30 minutes with uridine contain virus-specific RNA in the form of particles with a sedimentation coefficient 45S (the sedimentation coefficient of the complete virus equals 57S). The buoyant density of the RNP peak in CsCI equals 1.43 to 1.44 g/cm. Although these properties are compatible with the idea that they are informosome, the particles, and in particular their protein component, should be characterized in more detail before reaching a definite conclusion. Recently SOS virus RNA-containing particles with p = 1.40 g/cm have been found in HeLa cells infected with poliovirus (Huang and Baltimore, 1970), although the authors have some doubts about the reality of these complexes. 

Volkova, M. Y., V. M. Zaides, and V. G. Zaslavsky. 1969. Slowly sedimenting particles present in cytoplasmic extract of Ehrlich ascites cells infected by Sendai virus. Molec. Biol. (U.S.S.R.), 3 635-638. 

---