Phosphorylation controls autoinhibition of cytoplasmic linker protein-170

Author Posting. © American Society for Cell Biology, 2010. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 21 (2010): 2661-2673, doi:10.1091/mbc.E09-12-1036.Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.This work was supported by National Institutes of Health grant GM-25062 (to G.G.B.); Netherlands Organization for Scientific Research grants (to A. A. and N. G.); a Cancer Genomics Centre grant (to J.v.H.); and Presidential Program of Russian Academy of Sciences and RFBP grant 05-04-4915 (to E.S.N.)

Automated Acquisition of Cryo Electron Micrographs Using Leginon

Molecular microscopy is one of the most important structural approaches in cell biological investigations and can provide insight into complex biological questions that no other technique can provide. Currently, the technique typically requires the acquisition of very large numbers of transmission electron micrographs from frozen hydrated specimens using low dose techniques. The field is constrained by manual data acquisition methods that are slow, labor-intensive and result in a very low percentage of suitable images. We have developed a system, called Leginon, for automatically acquiring images from a transmission electron microscope. Our first prototype of this system demonstrated that we could acquire 1000 high magnification images per day from negatively stained catalase crystals. We have now extended this system to acquire low dose images of specimens embedded in vitreous ice.Methods: Specimens were prepared on Quantifoil grids using techniques which have been described previously. The Leginon system uses a Philips CM200 TEM and a Gatan MSC CCD camera and is controlled by the emScope software library. The overall acquisition protocol requires (i) obtaining a low magnification image [660x] of a grid square from a Quantifoil grid (fig. 1a); (ii) automatically identifying holes containing ice of suitable thickness; (iii) acquiring an intermediate magnification image [6600x] of the identified hole (fig. 1b); (iv) identifying features of interest within the hole; (v) focusing at high magnification [38,000x] and finally (vi) acquiring a high magnification image (fig 1c,d).</jats:p

An Integrated System for Transmission Electron Microscopy.

Macromolecular microscopy is becoming an increasingly important tool for structural biology. The development of improved capabilities for three-dimensional electron microscopy is critical for optimal progress in emerging integrative research in molecular cell biology. These techniques currently suffer from several severe disadvantages related to the tremendous time and effort required to acquire and analyze the data.For several years we have been developing software for automated and intelligent acquisition of transmission electron micrographs [1,2,3]. Our overall goal is to develop a system for rapid and routine structure determination of macromolecular assemblies from specimens preserved in vitreous ice. Ultimately we plan to develop an integrated system that can produce an electron density map of a structure within a few hours of inserting a specimen into the electron microscope. With this goal in mind it is essential that the images be acquired using a digital camera rather than film.</jats:p

Leginon: a system for fully automated acquisition of 1000 electron micrographs a day.

We have developed a system to automatically acquire large numbers of acceptable quality images from specimens of negatively stained catalase, a biological protein which forms crystals. In this paper we will describe the details of the system architecture and analyze the performance of the system as compared to a human operator. The ultimate goal of the system if to automate the process of acquiring cryo-electron micrographs