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Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail’s lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca2+signaling by inositol trisphosphate receptors (IP3R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca2+-mediated interactions between IP3R. Ca2+released by a cluster of IP3R (giving a local Ca2+puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca2+spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca2+puffs (interpuff intervals, IPI) and Ca2+spikes (interspike intervals) evoked by flash photolysis of caged IP3. We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca2+spikes do not arise from oscillatory dynamics of IP3R clusters, but that repetitive Ca2+spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.

The subcellular localization of membrane Ca2+channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca2+nanoscale resolution (SCCaNR), employing Ca2+-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca2+-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP3R) channels underlying local Ca2+puffs. Fluorescence signals (blips) arising from single functional IP3Rs are almost immotile (diffusion coefficient <0.003 μm2s−1), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP3Rs distributed within clusters that have an overall diameter of ∼400 nm, indicating that the nanoscale architecture of IP3R clusters is important in shaping local Ca2+signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca2+channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca2+signaling; however, our simulations indicate that passive diffusion of Ca2+ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP3)-evoked Ca2+signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca2+signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP3receptors along the TNT. We propose that IP3receptors may actively propagate intercellular Ca2+signals along TNTs via Ca2+-induced Ca2+release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.