Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t 1/2 ] of 4 to 6 min) of agonist-and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G 0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin ␣. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t 1/2 ‫؍‬ 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin ␣. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1 ؊ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.The predominant pathway for the nuclear import of transcription factors and other nuclear regulatory proteins originates with the interaction of importin ␣-like proteins (also called karyopherin ␣, Rch1/hSRP␣, hSRP1/NPI-1, and pendulin/OHO31) with specific nuclear localization sequences (NLSs), which contain closely spaced arrangements of five to eight basic amino acids (31,62,64). For DNA sequence-specific transcription factors, NLSs generally colocalize with their DNA binding domains (DBDs), which appears to reflect a coevolutionary selective pressure to ensure that proteins that bind DNA are able to access the nucleus (52). Nuclear export, by contrast, occurs through alternative pathways, which for many proteins involves the binding of CRM1 (exportin 1) to hydrophobic nuclear export sequences (26,90).However, some transcription factors, including the glucocorticoid hormone receptor (GR), contain additional NLSs that occur in other regions of the proteins (69,89,95,99). In at least some instances, the presence of these additional NLSs has been found to reflect a requirement for specialized or tightly regulated nuclear localization of the protein. For example, the nuclear localization potential of one of the two NLSs in the adenovirus E1A protein is active only during early development (92), while two of the three c-abl NLSs promote nuclear localization of c-abl only in certain cell types (97, 99). T...

Glucocorticoid receptor (GR) recycles between an inactive form complexed with heat shock proteins (hsps) and localized to the cytoplasm and a free liganded form that regulates specific gene transcription in the nucleus. We report here that, contrary to previous assumptions, association of GR into hsp-containing complexes is not sufficient to prevent the shuttling or trafficking of the GR across the nuclear membrane. Following the withdrawal of treatment with cortisol or the hormone antagonist RU486, GRs recycled rapidly into hsp-associated, hormone-responsive complexes. However, cortisolwithdrawn receptors redistributed to the cytoplasm very slowly (t1 ⁄2 ‫؍‬ 8 -9 h) and RU486-withdrawn receptors not at all. Persistent localization of these GRs to the nucleus was not due to a gross defect in export, since in both instances the complexed nuclear GRs transferred efficiently between heterokaryon nuclei. Moreover, the addition of a nuclear retention signal to the N terminus of GR induced the transfer of naive receptor to the nucleus in the absence of steroid. These results suggest that the localization of GR to the cytoplasm is determined by fine control of the rates of transfer of GR across the nuclear membrane and/or by active retention that occurs independently from the association of GR with hsps.

Glucocorticoid receptor (GR) exchanges between an active nuclear form and a complexed inactive, steroid-sensitive cytoplasmic form. Using a semi-quantitative indirect immunofluorescence assay to measure the kinetics of subcellular redistribution of GR in response to challenge during G(o), we have found that the ability to bind DNA is an important determinant for localization and tight binding of GR to the nucleus. The transfer of GR DNA-binding mutants to the nucleus after treatment with hormone agonists and antagonists was markedly reduced. Further, mutant receptors localized to the nucleus were only weakly associated with the nuclear compartment as they were released into cytosol upon hypotonic lysis of the cell membrane. Moreover, after agonist withdrawal, GR redistributed to the cytoplasm more rapidly when unable to bind DNA. By contrast, withdrawal of the hormone antagonist RU486 was found to yield a form of wild type GR that was completely unable to redistribute to the cytoplasm. However, this did not appear to result from a block in nuclear export as selective inactivation of nuclear import with energy inhibitor released RU486-withdrawn GRs from the nucleus at the same rates as agonist-withdrawn receptors. In addition, GR mutants unable to bind DNA, which retained a significant presence in the cytoplasm both during and after antagonist treatment, also failed to redistribute. The effect of RU486 treatment did not appear to be mediated through a block in reassociation of GR into a steroid-responsive form as RU486-withdrawn wild type receptors retained full potential to activate transcription from a glucocorticoid-responsive promoter after a second challenge with hormone. Therefore, reassociation of GR into a steroid-responsive form appears to be independent of signals important for the retention of GR in the cytoplasm.

A major obstacle in the development of microinjection technology for plant protoplasts has been the poor resolution of the intracellular compartments by conventional light microscopy. This was overcome through the use of fluorescent stains. The nuclei of living alfalfa (Medicago sativa) protoplasts were stained specifically with both Hoechst 33258 and 4′,6-diamidino-2-phenylindole. Mitochondria were stained specifically with 3,3′-diethyloxadicarbocyanine iodide and rhodamine 123. Cytoplasm was stained selectively with fluorescein diacetate. Under the specific conditions employed, the stains did not retard or suppress DNA synthesis, mitosis, cell division, or microcolony formation. Intranuclear microinjection did not significantly inhibit cell division in protoplasts stained with Hoechst 33258. Furthermore, the pattern of protein synthesis remained constant during the early stages of protoplast culture and was not perturbed by staining. These combined results demonstrated the suitability of alfalfa protoplasts stained with fluorescent dyes as hosts for microinjection.

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Procedures and equipment have been developed for the efficient intranuclear microinjection of plant protoplasts. The manipulation and culture of protoplasts was facilitated by the use of an environmentally controlled cabinet and video recording equipment. Microinjection rates of 80–100 nuclei/h were routine. The delivery of volumes as small as 10−17 L was easily controlled with a custom-designed microinjection syringe system. Individual protoplasts were recovered with 90% efficiency with the assistance of microscopie grid patterns photolithographically applied to cover slips.

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