Fusion pore expansion and contraction during catecholamine release from endocrine cells

ABSTRACTAmperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a pre-spike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicle’s densecore. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model recapitulated the rapid phases of a spike, but generated relations between spike shape parameters that differed from experimental results. To explore the possibility of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course. g/V initially tracks the pre-spike foot and the start of the spike, increasing ∼15-fold to the peak. However, after the spike peaks, g/V unexpectedly declines and settles to a constant value that indicates the presence of a stable post-spike pore. g/V of the post-spike pore varies greatly between events, and has an average that is ∼3.5-fold below the peak value and ∼4.5-fold above the pre-spike value. The post-spike pore persists and g/V remains flat for tens of milliseconds, as long as catecholamine flux can be detected. Applying the g/V transform to rare events with two peaks revealed a stepwise increase in g/V during the second peak. The g/V transform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a new framework for analyzing the actions of proteins that alter spike shape. The stable post-spike pore conforms with predictions from lipid bilayer elasticity, and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles.STATEMENT OF SIGNIFICANCEAmperometry recordings of catecholamine release from single vesicles reveal a complex waveform with distinct phases. The role of the fusion pore in this waveform is poorly understood. A model based on a static fusion pore fails to recapitulate important aspects of the waveform. A new transform of amperometric current introduced here renders fusion pore permeability in real time. This transform reveals rich dynamic behavior of the fusion pore as catecholamine leaves a vesicle. This analysis shows that fusion pore permeability rapidly increases and then decreases before settling into a stable post-spike configuration.

A Further Study on the Analytical Model for the Permeability in Flip-Chip Packaging

The notion of permeability is very important in understanding and modeling the flow behavior of fluids in a special type of porous medium (i.e., the underfill flow in flip-chip packaging). This paper presents a new concept regarding permeability in a porous medium, namely two types of permeability: superficial permeability (with consideration of both the pore cross-sectional area and the solid matrix cross-sectional area) and pore permeability (with consideration of the pore cross-sectional area only). Subsequently, the paper proposes an analytical model (i.e., equation) for the pore permeability and superficial permeability of an underfill porous medium in a flip-chip packaging, respectively. The proposed model along with several similar models in literature is compared with a reliable numerical model developed with the computational fluid dynamics (CFD) technique, and the result of the comparison shows that the proposed model for permeability is the most accurate one among all the analytical models in literature. The main contributions of the paper are as follows: (1) the provision of a more accurate analytical model for the permeability of an underfill porous medium in flip-chip packaging, (2) the finding of two types of permeability depending on how the cross-sectional area is taken, and (3) the correction of an error in the others' model in literature.

TP53 exon-6 truncating mutations produce separation of function isoforms with pro-tumorigenic functions

TP53 truncating mutations are common in human tumors and are thought to give rise to p53-null alleles. Here, we show that TP53 exon-6 truncating mutations occur at higher than expected frequencies and produce proteins that lack canonical p53 tumor suppressor activities but promote cancer cell proliferation, survival, and metastasis. Functionally and molecularly, these p53 mutants resemble the naturally occurring alternative p53 splice variant, p53-psi. Accordingly, these mutants can localize to the mitochondria where they promote tumor phenotypes by binding and activating the mitochondria inner pore permeability regulator, Cyclophilin D (CypD). Together, our studies reveal that TP53 exon-6 truncating mutations, contrary to current beliefs, act beyond p53 loss to promote tumorigenesis, and could inform the development of strategies to target cancers driven by these prevalent mutations.

Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of β-Catenin in Zebrafish

Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. Thenup62lgene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulatesnup62ltranscription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulatenup62lexpression. Overexpression ofnup62ldorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish.

Protein diffusion through charged nanopores with different radii at low ionic strength

Pore permeability for two similar molecular weight proteins (BSA and BHb) through nanoporous charged membranes at low ionic strength (I = 0.001 M).