Exchange of information between the nucleus and cytosol depends on the metabolic state of the cell, yet the energy-supply pathways to the nuclear compartment are unknown. Here, the energetics of nucleocytoplasmic communication was determined by imaging import of a constitutive nuclear protein histone H1. Translocation of H1 through nuclear pores in cardiac cells relied on ATP supplied by mitochondrial oxidative phosphorylation, but not by glycolysis. Although mitochondria clustered around the nucleus, reducing the distance for energy transfer, simple nucleotide diffusion was insufficient to meet the energetic demands of nuclear transport. Rather, the integrated phosphotransfer network was required for delivery of high-energy phosphoryls from mitochondria to the nucleus. In neonatal cardiomyocytes with low creatine kinase activity, inhibition of adenylate kinase-catalyzed phosphotransfer abolished nuclear import. With deficient adenylate kinase, nucleoside diphosphate kinase, which secures phosphoryl exchange between ATP and GTP, was unable to sustain nuclear import. Up-regulation of creatine kinase phosphotransfer, to mimic metabolic conditions of adult cardiac cells, rescued H1 import, suggesting a developmental plasticity of the cellular energetic system. Thus, mitochondrial oxidative phosphorylation coupled with phosphotransfer relays provides an efficient energetic unit in support of nuclear transport. E fficient communication between the cytosol and nucleus is essential in cellular homeostasis, regulating proper processing of genetic and metabolic information. Central in nucleocytoplasmic exchange is the transport of macromolecules across the nuclear envelope (1, 2), a multistep process that initially proceeds by signal-mediated recognition of the macromolecule to be transported, following by docking events and, ultimately, translocation through nuclear pores (1-5). In energy-depleted cells, molecules that are actively transported into the nucleus, such as the constitutive chromatin protein histone H1, tend to accumulate on the cytosolic surface of the nuclear membrane (2, 4). While formation and docking of the transported protein, complexed with a transport receptor, may be energy-independent, the actual translocation and accumulation of molecules in the nuclear compartment against a concentration gradient may, however, require an energy source (4).Energy-consuming enzymes, including nucleoside triphosphatases, are associated with the nuclear envelope, and their activity is stimulated in the presence of the transported substrate (6, 7). Underscoring the energetic cost of nuclear transport, receptor cycling and continued signal processing mandate catalytic conversion of the guanine nucleotide-binding protein Ran from Ran-GTP to Ran-GDP, which is accomplished by the RanGTPase and the subsequent regeneration of GTP (1, 2). Yet, transport of macromolecules across the nuclear envelope that can proceed in an apparently energy-independent manner also has been reported (8, 9). This observation was, however, made i...

Abstract-Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca 2ϩ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca 2ϩ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca 2ϩ homeostasis. (Circ Res. 1999;84:1292-1301.)

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is ''downstream'' of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival. The primary and potentially reversible steps of toxicity mediated by mutant Huntington's disease (HD) protein [mutant huntingtin (mhtt)] are controversial. A major model suggests that short cleavage products of mhtt migrate to the nucleus and cause transcriptional dysfunction (1). The N-terminal truncated form of mhtt is known to bind and interfere with nuclear factors such as cAMP response element-binding protein (CREB) (2), CREB-binding protein (3), corepressor (4), and transcriptional activator Sp1 (5). Restoration of transcription by histone deacetylase inhibitors improves survival of affected neurons in a Drosophila model for HD (6), indicating that mhtt can cause deleterious alterations in transcription. However, transcriptional dysfunction is caused in cell, mouse, or fly models by expression of only the short truncated fragment of the mhtt (6-10). Short fragments can freely migrate into the nucleus and cause dysfunction there (10). In vivo, however, fragments must arise from cleavage of full-length mhtt, a large cytoplasmic protein with no nuclear localization signal (NLS) (10). Yet neither the timing nor the extent of cleavage of the full-length mhtt in vivo is known. Recent data suggest that cleavage of mhtt may be slow in human HD tissue (11) and raise the possibility that N-terminal cleavage may not be an early or primary event in toxicity.In fact, several lines of evidence suggest that nuclear events may not be sufficient to account for the initial toxic effects of mhtt. In presymptomatic or late-onset disease tissue, nuclear inclusions (NI) are often absent in patients, whereas cytoplasmic inclusions can exceed NIs (12). Second, many mhtt-interacting proteins identified by yeast two-hybrid are cytoplasmic proteins (13). Microarray analysis of HD mice reveals that expression of many trafficking proteins and cytoplasmic metabolizing enzymes is affected early in the toxic progression (14). Moreover, Sp1 has been shown to be a major target of mhtt, presumably mediating toxicity by repression of Sp1-dependent genes (15). However, many Sp1-dependent genes are unaffected by exp...

Each nuclear pore is responsible for both nuclear import and export with a finite capacity for bidirectional transport across the nuclear envelope. It remains poorly understood how the nuclear transport pathway responds to increased demands for nucleocytoplasmic communication. A case in point is cellular hypertrophy in which increased amounts of genetic material need to be transported from the nucleus to the cytosol. Here, we report an adaptive down-regulation of nuclear import supporting such an increased demand for nuclear export. The induction of cardiac cell hypertrophy by phenylephrine or angiotensin II inhibited the nuclear translocation of H1 histones. The removal of hypertrophic stimuli reversed the hypertrophic phenotype and restored nuclear import. Moreover, the inhibition of nuclear export by leptomycin B rescued import. Hypertrophic reprogramming increased the intracellular GTP/GDP ratio and promoted the nuclear redistribution of the GTP-binding transport factor Ran, favoring export over import. Further, in hypertrophy, the reduced creatine kinase and adenylate kinase activities limited energy delivery to the nuclear pore. The reduction of activities was associated with the closure of the cytoplasmic phase of the nuclear pore preventing import at the translocation step. Thus, to overcome the limited capacity for nucleocytoplasmic transport, cells requiring increased nuclear export regulate the nuclear transport pathway by undergoing a metabolic and structural restriction of nuclear import.Hypertrophy is a fundamental adaptive process that enables heart muscle to accommodate demands for increased workload or to compensate for the loss of cardiac cells (1-3). Hypertrophied cardiomyocytes display a distinct pattern of gene expression, increased content of contractile proteins, and augmented myofibrillogenesis (1-8). Such critical processes in hypertrophy depend on molecules that have to be carried into or out of the nucleus (9, 10). In particular, the amount of mRNAs that need to be transported from the nucleus to the cytosol and transcribed into proteins dramatically increases (1-3, 11-13). Considering the limited total capacity for nuclear transport (14 -17), it remains unknown how the nuclear transport pathway operates to support increased demands for nucleocytoplasmic communication.The nuclear envelope, which separates the nuclear content from the cytoplasm, mediates the transport required for the regulation of gene expression and processing of genetic information (14 -16). Although several steps in the process are recognized including targeting and movement to the nuclear surface, it is translocation through the nuclear envelope that ultimately secures the transfer of molecules (17-23). Translocation occurs through nuclear pore complexes, which span the nuclear envelope and gate bidirectional nucleocytoplasmic exchange (24 -26). In response to changes in cellular bioenergetics or ion homeostasis, nuclear pores adopt distinct conformations regulating nuclear import (24 -29). Transport can be activat...

Intracellular Ca2+ is released from intracellular stores in the endoplasmic reticulum (ER) in response to the second messenger inositol (1,4,5) trisphosphate (InsP3) [1,2]. Then, a poorly understood cellular mechanism, termed capacitative Ca2+ entry, is activated [3,4]; this permits Ca2+ to enter cells through Ca(2+)-selective Ca(2+)-release-activated ion channels [5,6] as well as through less selective store-operated channels [7]. The level of stored Ca2+ is sensed by Ca(2+)-permeant channels in the plasma membrane, but the identity of these channels, and the link between them and Ca2+ stores, remain unknown. It has been argued that either a diffusible second messenger (Ca2+ influx factor; CIF) [8] or a physical link [9,10] connects the ER Ca(2+)-release channel and store-operated channels; strong evidence for either mechanism is lacking, however [7,10]. Petersen and Berridge [11] showed that activation of the lysophosphatidic acid receptor in a restricted region of the oocyte membrane results in stimulation of Ca2+ influx only in that region, and concluded that a diffusible messenger was unlikely. To investigate the relationship between ER stores and Ca2+ influx, we used centrifugation to redistribute into specific layers the organelles inside intact Xenopus laevis oocytes, and used laser scanning confocal microscopy with the two-photon technique to 'uncage' InsP3 while recording intracellular Ca2+ concentration. Ca2+ release was localized to the stratified ER layer and Ca2+ entry to regions of the membrane directly adjacent to this layer. We conclude that Ca2+ depletion and entry colocalize to the ER and that the mechanism linking Ca2+ stores to Ca2+ entry is similarly locally constrained.