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.)

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

CLASP Modulates Microtubule-Cortex Interaction during Self-Organization of Acentrosomal Microtubules

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Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell–cell or cell–extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus–centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes

CK1 activates minus-end–directed transport of membrane organelles along microtubules

This study shows that the signal transduction pathway responsible for the initiation of minus-end–directed movement of membrane-bounded pigment granules in melanophores involves sequential activation of protein phosphatase 2A and casein kinase 1 and that this activation correlates with increased phosphorylation of the dynein intermediate chain