Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.

Chlorophyll Synthesis in Dark-Grown Pine Primary Needles1

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide1

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.

Different Phototransduction Kinetics of Phytochrome A and Phytochrome B in Arabidopsis thaliana1

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of β-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h−1 (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h−1 caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.

Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.

The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.

Kinetics of spindle pole body separation in budding yeast.

In the budding yeast Saccharomyces cerevisiae, the spindle pole body (SPB) serves as the microtubule-organizing center and is the functional analog of the centrosome of higher organisms. By expressing a fusion of a yeast SPB-associated protein to the Aequorea victoria green fluorescent protein, the movement of the SPBs in living yeast cells undergoing mitosis was observed by fluorescence microscopy. The ability to visualize SPBs in vivo has revealed previously unidentified mitotic events. During anaphase, the mitotic spindle has four sequential activities: alignment at the mother-daughter junction, fast elongation, translocation into the bud, and slow elongation. These results indicate that distinct forces act upon the spindle at different times during anaphase.