Protein loss during nuclear isolation.

Abstract

Cryomicrodissection makes possible the measurement of the entire in vivo protein content of the amphibian oocyte nucleus and provides a heretofore missing baseline for estimating protein loss during nuclear isolation by other methods. When oocyte nuclei are isolated into an aqueous medium, they lose 95% of their protein with a half-time of 250 s. This result implies an even more rapid loss of protein from aqueously isolated nuclei of ordinary-size cells.Cell nuclei are isolated in aqueous media in many laboratories, and their analyses are used to characterize structures and functions of the in vivo nucleus. Because the nuclear envelope contains pores permeable to macromolecules (1-4), it is understood that some proteins must be lost (5). However, the magnitude of the loss is unknown, because the in vivo (preisolation) protein content of nuclei has not been determined and compared to the protein remaining in isolated nuclei. We present here a two-step approach to this problem. First, we determined the in vivo protein content of the large nucleus (400-500-~m diameter) of the amphibian oocyte isolated by cryomicrodissection. Second, with this in vivo content as a baseline, we measured the kinetics of protein loss from oocyte nuclei isolated directly into an aqueous medium. MATERIALS AND METHODSCryomicrodissection (6, 7) is a method in which individual oocytes are frozen in liquid nitrogen and subsequently maintained at less than -45"C while the nucleus is microsurgically isolated with fine-tipped stainless steel microtools (Fig. I). The low temperature prevents diffusive relocations of nuclear and cytoplasmic solutes from their in vivo locations. Clean, intact nuclei cyomicrodissected from Xenopus oocytes (stages V and VI) (8) varied somewhat in wet weight from animal to animal, but their size distribution was narrow for cells from the same animal (standard error of the mean <5%). Nuclear water contents, determined from wet and dry weights of cryomicrodissected nuclei, were relatively constant (even between animals) at 87.2 +_ 0.3%, with the dry mass consisting almost entirely of protein. The nuclei of the oocytes from the two animals used in the present study had protein contents of 3.8 _ 0.4 and 5.5 _+ 0.7 ug (Fig. 2, upper and lower curves, respectively.)To isolate nuclei into aqueous solution, we punctured and compressed individual oocytes with forceps (9, 10) until the nucleus was extruded. The medium was Ca2÷-free and formulated (legend, Fig. 2) to mimic the oocyte's intracellular free monovalent cation concentrations (7). After extrusion, each nucleus was gently pipetted through the medium to remove traces of adherent cytoplasm, incubated without agitation in fresh medium for time t~, and assayed for protein content. Nonspherical (damaged) nuclei were discarded. RESULTS AND DISCUSSIONThe aqueous isolation procedure we used is gentle compared to the mass cell shearing or homogenization employed in most studies. Nevertheless, even under these conditions, 1240 loss of nuclear protein was 90% by 1 h, an...

A Pathway for the Control of Anoikis Sensitivity by E-Cadherin and Epithelial-to-Mesenchymal Transition

“…S10 in the supplemental material). Nuclear versus cytoplasmic localization of NRAGE could not be shown reliably using cell fractionation methods, which is consistent with previous observations that some nucleoplasmic proteins tend to leak out of the nucleus during fractionation, precluding this experimental approach (12,25,69). Nevertheless, the shift toward nuclear NRAGE in EMT-derived cells or in normal cells depleted of ankyrin-G, or, conversely, the shift toward cytoplasmic NRAGE upon the add-back of ankyrin-G to EMT-derived cells strongly implicated the loss of ankyrin-G in the EMT-driven NRAGE relocalization.…”

Diffusive and nondiffusive proteins in vivo.

“…Our value for nuclear actin, 0.20 kg, is somewhat larger than the values obtained in previous determinations performed on aqueously isolated oocyte nuclei (32,33). However, the difference, 0 .05-0.07 gg, is similar to the amount of actin that we calculate to be diffusive within the in vivo nucleus (see below), which is consistent with diffusive protein loss from the aqueously isolated oocyte nucleus (8).…”

“…These parameters cannot be measured by aqueous methodologies because both are sensitive to the composition and ordering ofthe in vivo aqueous milieu : uncontrolled changes in these parameters occur during cell disruption (7,8) and also must be suspected of accompanying chemical fixation and cell permeabilization . By application of techniques designed to obviate these difficulties, we have sought to determine protein spatial and biochemical relationships lost by other methods and to better understand protein functions within the living cell.…”

The Nck SH2/SH3 adaptor protein is present in the nucleus and associates with the nuclear protein SAM68

“…We have demonstrated that Nck is both a nuclear and a cytoplasmic protein and that this subcellular location is independent of growth factor stimulation by both confocal microscopy and Western blot analysis of subcellular fractions. The amount of Nck in the nucleus could be substantial, but as with the quantitation of MAP kinase (Chen et al, 1992) a more precise determination is dicult due to the signi®cant amount of protein leakage from nuclei during cell fractionation (Paine et al, 1983). Since Nck does not contain a known nuclear localization signal (NLS), its presence in the nucleus may be through its association with other proteins.…”