Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.
Abstract:The integration of Building Information Modeling (BIM) and Geographic Information System (GIS) has been identified as a promising but challenging topic to transform information towards the generation of knowledge and intelligence. Achievement of integrating these two concepts and enabling technologies will have a significant impact on solving problems in the civil, building and infrastructure sectors. However, since GIS and BIM were originally developed for different purposes, numerous challenges are being encountered for the integration. To better understand these two different domains, this paper reviews the development and dissimilarities of GIS and BIM, the existing integration methods, and investigates their potential in various applications. This study shows that the integration methods are developed for various reasons and aim to solve different problems. The parameters influencing the choice can be summarized and named as "EEEF" criteria: effectiveness, extensibility, effort, and flexibility. Compared with other methods, semantic web technologies provide a promising and generalized integration solution. However, the biggest challenges of this method are the large efforts required at early stage and the isolated development of ontologies within one particular domain. The isolation problem also applies to other methods. Therefore, openness is the key of the success of BIM and GIS integration.
We report that, during epiboly in zebrafish, three F-actin-based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring-like F-actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo-BAPTA results in the disruption of all three of these actin-based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca 2ϩ , as well as the functions of other previously described F-actin-based structures observed throughout epiboly, are discussed. Developmental Dynamics 231: 313-323, 2004.
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