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FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.

ABSTRACT: The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo-cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than approximately 30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation-promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin-mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess 'self-healing' properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.

SUBMITTER: Frey S

PROVIDER: S-EPMC2728434 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.

Frey Steffen S Görlich Dirk D

The EMBO journal 20090813 17

The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo-cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than approximately 30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation-promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repe ...[more]

PMID: 19680227

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Project description:Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm2 on day 3 on chip and were sustained above 2000 Ω · cm2 up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc.

Project description:Rationale. The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/b-catenin signaling is responsible for the early phases of brain vascularization and blood-brain barrier differentiation. However, this signal declines after birth and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. Objective. Sox17 constitutes a major downstream target of Wnt/b-catenin in endothelial cells and regulates arterial differentiation. In the present paper, we asked whether Sox17 may act downstream of Wnt/b-catenin in inducing BBB differentiation and maintenance. Methods and Results. Using reporter mice and nuclear staining of Sox17 and b-catenin, we report that while b-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo, and progressively declines after birth. In search of Sox17 mechanism of action, RNA-Seq analysis of gene expression of brain endothelial cells has identified members of the Wnt/b-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/b-catenin signaling and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the b-catenin destruction complex or expression of a degradation-resistant b-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. Conclusions. Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB and they underline the strict reciprocal tuning of this transcription factor and Wnt/b-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

Dataset Information

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.

Publications

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self-healing properties.

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