9/22/2023 0 Comments Scidavis emission spectrum![]() An IgG binding purification tag (ZZ) was fused to the N-terminus of MZT2 ( Fig. Encouraging yields were only obtained when a stoichiometric excess of γ-tubulin, GCP2, and GCP3 were co-overexpressed in insect cells with GCP4, GCP5, GCP6, NEDD1, MZT1, β-actin, and MZT2A (hereafter, MZT2 Wieczorek et al., 2020a ). We tested multiple strategies, including expressing various combinations of γ-TuRC components either in human cells under cytomegalovirus promoters or in insect cells using the baculovirus expression system. To address the need for a biochemically tractable system with which to study the function and assembly of a vertebrate γ-TuRC, we asked whether the human complex could be reconstituted with recombinant proteins. However, the role of the LB in the assembly and microtubule-nucleating activity of the γ-TuRC is currently unclear. The LB is composed of two copies of MZT1 that associate with the N-terminal α-helical domains (NHDs) of GCP3 and GCP6 in structurally mimetic “modules,” along with an actin-like protein ( Wieczorek et al., 2020a). Unexpectedly, the γ-TuRC also contains a prominent feature inside its conical structure, termed the “lumenal bridge” (LB). The γ-TuRC is an asymmetric, cone-shaped structure, in which GCP2-6 orient 14 γ-tubulin molecules in a helical arrangement that is poised to nucleate microtubules. The stoichiometry, location, and structures of these proteins in the context of the native human γ-TuRC were recently identified using high-resolution cryo-EM ( Wieczorek et al., 2020a, b Consolati et al., 2020). In vertebrates, the γ-TuRC contains at least 8 proteins, including γ-tubulin, the γ-tubulin complex proteins (GCPs) 2–6, and the mitotic spindle organizing proteins associated with a ring of γ-tubulin-1 and -2 (MZT1 and MZT2 Murphy et al., 2001 Teixidó-Travesa et al., 2010 Hutchins et al., 2010). The γ-tubulin ring complex (γ-TuRC) is an ∼2.3 MD assembly required for proper microtubule network formation in eukaryotes ( Knop et al., 1997 Raff et al., 1993 Stearns and Kirschner, 1994 Zheng et al., 1995). Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRC ΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. We show that γ-TuRC ΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. In addition, we generate a subcomplex, γ-TuRC ΔLB-GFP, which lacks MZT1 and actin. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). More information, including screenshots, reviews, current contributors can be found on the SourceForge project webpage.The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). Particularly, this means that we will try to provide good documentation on all levels, ranging from user’s manual over tutorials down to and including documentation of the internal APIs We encourage users to share their experiences on our forums and on our mailing lists. What sets SciDAVis apart from the above is its emphasis on providing a friendly and open environment (in the software as well as the project) for new and experienced users alike. SciDAVis is similar in its field of application to proprietary Windows applications like Origin and SigmaPlot as well as free applications like QtiPlot, Labplot and Gnuplot. SciDAVis runs on GNU/Linux, Windows and MacOS X possibly also on other platforms like *BSD, although this is untested. It combines a shallow learning curve and an intuitive, easy-to-use graphical user interface with powerful features such as scriptability and extensibility. ![]() SciDAVis is a free interactive application aimed at data analysis and publication-quality plotting.
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