Structural, functional, and conceptual views of native architecture in the Xenopus oocyte. (a) Punctate immunolocalization of native clusters of in the cortical ER of a Xenopus oocyte resolved by immunofluorescence. (b) Top: linescan image of two discrete puffs resolved using fluo-4 ( for ) that were evoked by photorelease of . Bottom: associated fluorescence profiles. (c) Schematic model, combining (a) and (b), conceptualizing puffs as the local elementary release occurring from discrete clusters (cubes) spaced throughout the ER. Summation of the stochastic behavior of these elementary events is required to trigger a wave and establish repetitive oscillations. puffs occur independently at low levels of stimulation, but activity is coordinated by during maintained stimulation ( -axis) to trigger a global whole-cell signal. This summation is often coordinated by the activity of particular “focal” puffs sites (dark gray) that serve as the spatial focus of wave initiation. In the Xenopus oocyte, the amount of liberated by a puff is well below the threshold to initiate a wave, allowing the coexistence of local and global signaling modalities in the same cell.
Functional architecture of —structural and functional distinction of native clusters and activity-induced aggregates. socialization from (i) “unitary” channels to (ii) native clusters that support puffs to (iii) larger aggregates observed following sustained stimulation. While little is known about mechanisms regulating cluster assembly, a change in conformation (e.g., diamond to hourglass) is a prerequisite for aggregate formation. The functional effects of socialization may not scale linearly with channel number, rather different architectures reflect distinct states of activity: native “loose” clustering promotes activity, whereas high density packing or activity-induced aggregation attenuates activity. Numerical estimates are from Refs. 45, 56, and 101 . For clarity, individual tetramers are represented in different shades of gray.
mobility within the ER. Examples of FRAP-derived estimates for the diffusion coefficient and mobile fraction of fluorescent-protein tagged in different cell types. Please refer to references for details of how these parameters were calculated in individual studies ( reported).
Activity-induced clustering of in various cell lines. Summary of studies investigating the formation and properties of clusters evoked by cellular stimulation. Readers are referred to indicated references for further experimental detail. Exogenous constructs were expressed by transient transfection, unless indicated otherwise.
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