Calcium release and influx colocalize to the endoplasmic reticulum.

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.

Structural plasticity of the cardiac nuclear pore complex in response to regulators of nuclear import.

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 Ca2+ 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 Ca2+ 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 Ca2+ homeostasis.

Directed inhibition of nuclear import in cellular hypertrophy.

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.