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Houston, TX, United States

Narayanan A.S.,Verna and Marrs McLean | Reyes S.B.,University of Texas M. D. Anderson Cancer Center | Um K.,Baylor College of Medicine | McCarty J.H.,University of Texas M. D. Anderson Cancer Center | And 2 more authors.
Molecular Biology of the Cell | Year: 2013

Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCC) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-Activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCC signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCC activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCC function.© 2013 Shen and Osmani. This article is distributed by The American Society for Cell Biology under license from the author(s).

Venken K.J.T.,Verna and Marrs McLean | Venken K.J.T.,Baylor College of Medicine | Sarrion-Perdigones A.,Verna and Marrs McLean | Vandeventer P.J.,Verna and Marrs McLean | And 3 more authors.
Wiley Interdisciplinary Reviews: Developmental Biology | Year: 2016

A central challenge in investigating biological phenomena is the development of techniques to modify genomic DNA with nucleotide precision that can be transmitted through the germ line. Recent years have brought a boon in these technologies, now collectively known as genome engineering. Defined genomic manipulations at the nucleotide level enable a variety of reverse engineering paradigms, providing new opportunities to interrogate diverse biological functions. These genetic modifications include controlled removal, insertion, and substitution of genetic fragments, both small and large. Small fragments up to a few kilobases (e.g., single nucleotide mutations, small deletions, or gene tagging at single or multiple gene loci) to large fragments up to megabase resolution can be manipulated at single loci to create deletions, duplications, inversions, or translocations of substantial sections of whole chromosome arms. A specialized substitution of chromosomal portions that presumably are functionally orthologous between different organisms through syntenic replacement, can provide proof of evolutionary conservation between regulatory sequences. Large transgenes containing endogenous or synthetic DNA can be integrated at defined genomic locations, permitting an alternative proof of evolutionary conservation, and sophisticated transgenes can be used to interrogate biological phenomena. Precision engineering can additionally be used to manipulate the genomes of organelles (e.g., mitochondria). Novel genome engineering paradigms are often accelerated in existing, easily genetically tractable model organisms, primarily because these paradigms can be integrated in a rigorous, existing technology foundation. The Drosophila melanogaster fly model is ideal for these types of studies. Due to its small genome size, having just four chromosomes, the vast amount of cutting-edge genetic technologies, and its short life-cycle and inexpensive maintenance requirements, the fly is exceptionally amenable to complex genetic analysis using advanced genome engineering. Thus, highly sophisticated methods developed in the fly model can be used in nearly any sequenced organism. Here, we summarize different ways to perform precise inheritable genome engineering using integrases, recombinases, and DNA nucleases in the D. melanogaster. © 2016 Wiley Periodicals, Inc.

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