Nucleocytoplasmic transport: a thermodynamic mechanism.

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from, the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.

Protein dynamics in individual human cells: experiment and theory.

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

Diffusion of anionic and neutral GFP derivatives through plasmodesmata in epidermal cells of Nicotiana benthamiana.

Plasmodesmata (Pd) are trans-wall membrane channels that permit cell-to-cell transport of metabolites and other small molecules, proteins, RNAs, and signaling molecules. The transport of cytoplasmic soluble macromolecules is a function of the electrochemical gradient between adjacent cells, the number of Pd per interface between adjacent cells, Stokes radius (R(S)), area of the cytoplasmic annulus, and channel length. The size of the largest molecule that can pass through Pd defines the Pd size exclusion limit. However, since the shape and size of a molecule determines its capacity to diffuse through pores or tubes, R(S) is a better measure. Relatively small changes in R(S) can cause large differences in the mobility of molecular probes, particularly if the pore size is close to that of the probe. In addition, as the dimensions of a macromolecule approach that of the channel, membrane charge effects may become important. We employed quantitative tools and molecular modeling to measure the apparent coefficient of conductivity of Pd, C(Pd), for the non-targeted transport of macromolecules. This method allowed us to examine the influence of protein charge and R(S) on C(Pd) in Nicotiana benthamiana. The C(Pd) of modified green fluorescent proteins (GFPs) of different sizes but with the same charge as native GFP and of a more negatively charged derivative were determined. We found that the C(Pd) of cytoplasmic soluble GFP and cytoplasmic forms of modified GFP were the most strongly correlated with R(S) and that the apparent aberrant increase in C(Pd) of a negatively charged GFP derivative was, at least in part, the result of the charge effect on R(S).

Reversibility in nucleocytoplasmic transport.

Nucleocytoplasmic exchange of proteins and RNAs is mediated by receptors that usher their cargo through the nuclear pores. Peptide localization signals on each cargo determine the receptors with which it will interact. Those interactions are normally regulated by the small GTPase Ran. Hydrolysis of GTP provides the chemical energy required to create a bona fide thermodynamic pump that selectively and directionally accumulates its substrates across the nuclear envelope. A common perception is that cargo delivery is irreversible, e.g., a protein imported to the nucleus does not return to the cytoplasm except perhaps via a specific export receptor. Quantitative measurements using cell-free nuclei reconstituted in Xenopus egg extract show that nuclear accumulation follows first-order kinetics and reaches steady state at a level that follows a Michaelis-Menten function of the cytoplasmic cargo concentration. This saturation suggests that receptor-mediated translocation across the nuclear pore occurs bidirectionally. The reversibility of accumulation was demonstrated directly by exchange of the cytosolic medium and by fluorescence recovery after photobleaching. Based on our results, we offer a simple biophysical model that predicts the observed behavior. A far-reaching consequence is that the nuclear localization signal dictates the fate of a protein population rather than that of the individual molecules that bear it, which remain free to shuttle back and forth. This implies an open communication between the nucleus and cytoplasm and a ubiquitous mechanism for signaling in both directions.