Fertilization of a mammalian egg induces a series of ‘zinc sparks’ that are necessary for inducing the egg-to-embryo transition. Despite the importance of these zinc efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches to resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy dispersive spectroscopy, X-ray fluorescence microscopy, and 3D elemental tomography for high resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 106 zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.
SUMMARY Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step towards chromatin condensation during erythropoiesis in mice.
Learning how to assemble inorganic nanoparticles into ordered lattices may prove to be important for applications, such as, electronics, photonics, and catalysis. [1] Indeed, theoretical studies have shown that certain types of crystalline arrays of nanoparticles could potentially be used to generate photonic band-gap materials, negative index materials, and metamaterials at visible and infrared length scales. [2,3] The vast majority of work in this area has focused on the assembly of spherical particles. However, anisotropic nanoparticles, which display rich assembly behavior owing to their reduced symmetry, and have unique physical properties that can be engineered by controlling interparticle spacing and orientation, may provide access to even more interesting materials. [4][5][6][7][8] Moreover, they require design rules for predicting the way such nanoparticles can assemble and the types of structures that may be realized. The rapidly expanding library of available anisotropic nanoparticle building blocks provides exciting new opportunities to study colloidal assembly as a function of particle shape. [9][10][11] Herein, we introduce a directional entropic force approach (DEFA) for controlling the assembly of anisotropic nanoparticles into crystalline lattices. The method relies on surfactant micelle-induced depletion interactions [12] to assemble anisotropic gold nanoparticles into reconfigurable, nonclose-packed (open) superlattices in solution. The anisotropic nanoparticles align along their flat facets to maximize entropy, and therefore minimize the free energy of the system, leading to assemblies with long-range order. [13,14] Importantly, our experimental work complements recent theoretical work that proposes directional entropic forces between nanoparticle facets as a viable means for thermodynamically assembling nanoparticle superlattices. [14][15][16][17] The experimental work herein uses depletants to create strong attractive forces that can drive assembly of reversible superlattices with tunable spacing in solution. These directional entropic forces are analogous to the directional bonding between atoms in molecules. [14,15,18] The resulting crystalline superlattices are therefore shape-dependent. We show that the electrostatic and depletion interactions combine to determine the lattice spacing, and can be tuned independently with surfactant concentration and ionic strength to reconfigure the lattice constant.The DEFA presented herein complements several assembly strategies that have been developed to create superlattices of anisotropic nanostructures including evaporationinduced [19,20] and sedimentation-based methods, [8] as well as ones based upon the manipulation of electrostatic, [21,22] entropic, [13,[23][24][25][26][27][28] block copolymer, [29,30] and biological recognition interactions. [31] However, unlike the majority of these methods, our approach yields reversible crystals with tunable lattice constants in situ.Depletion interactions are purely entropic in nature and arise when non-ads...
During fertilization or chemically-induced egg activation, the mouse egg releases billions of zinc atoms in brief bursts known as ‘zinc sparks.’ The zona pellucida (ZP), a glycoprotein matrix surrounding the egg, is the first structure zinc ions encounter as they diffuse away from the plasma membrane. Following fertilization, the ZP undergoes changes described as ‘hardening’, which prevent multiple sperm from fertilizing the egg and thereby establish a block to polyspermy. A major event in zona hardening is cleavage of ZP2 proteins by ovastacin; however, the overall physiochemical changes contributing to zona hardening are not well understood. Using x-ray fluorescence microscopy, transmission and scanning electron microscopy, and biological function assays, we tested the hypothesis that zinc release contributes to ZP hardening. We found that the zinc content in the ZP increases by 300% following activation and that zinc exposure modulates the architecture of the ZP matrix. Importantly, zinc-induced structural changes of the ZP have a direct biological consequence; namely, they reduce the ability of sperm to bind to the ZP. These results provide a paradigm-shifting model in which fertilization-induced zinc sparks contribute to the polyspermy block by altering conformations of the ZP matrix. This adds a previously unrecognized factor, namely zinc, to the process of ZP hardening.
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