Cytoplasmic linker proteins promote microtubule rescue in vivo

The role of plus end–tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin

Histone Demethylases KDM4A and KDM4C Regulate Differentiation of Embryonic Stem Cells to Endothelial Cells

SummaryUnderstanding epigenetic mechanisms regulating embryonic stem cell (ESC) differentiation to endothelial cells may lead to increased efficiency of generation of vessel wall endothelial cells needed for vascular engineering. Here we demonstrated that the histone demethylases KDM4A and KDM4C played an indispensable but independent role in mediating the expression of fetal liver kinase (Flk)1 and VE-cadherin, respectively, and thereby the transition of mouse ESCs (mESCs) to endothelial cells. KDM4A was shown to bind to histones associated with the Flk1 promoter and KDM4C to bind to histones associated with the VE-cadherin promoter. KDM4A and KDM4C were also both required for capillary tube formation and vasculogenesis in mice. We observed in zebrafish that KDM4A depletion induced more severe vasculogenesis defects than KDM4C depletion, reflecting the early involvement of KDM4A in specifying endothelial cell fate. These findings together demonstrate the essential role of KDM4A and KDM4C in orchestrating mESC differentiation to endothelial cells through the activation of Flk1 and VE-cadherin promoters, respectively

Migration and actin protrusion in melanoma cells are regulated by EB1 protein

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Cancer Letters 284 (2009): 30-36, doi:10.1016/j.canlet.2009.04.007.Remodeling of actin and microtubule cytoskeletons is thought to be coupled; however, the interplay between these two systems is not fully understood. We show a microtubule end-binding protein, EB1, is required for formation of polarized morphology and motility of melanoma cells. EB1 depletion decreased lamellipodia protrusion, and resulted in loss of opposed protruding and retracting cell edges. Lamellipodia attenuation correlated with mis-localization of filopodia throughout the cell and decreased Arp3 localization. EB1-depleted cells displayed less persistent migration and reduced velocity in singlecell motility experiments. We propose EB1 coordinates melanoma cell migration through regulating the balance between lamellipodial and filopodial protrusion.This work was supported by American Heart Association grant 0525660Z (J.M.S.) and Southern Illinois University Edwardsville FUR grant

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.)

Analysis of a microscopic stochastic model of microtubule dynamic instability

A novel theoretical model of dynamic instability of a system of linear (1D) microtubules (MTs) in a bounded domain is introduced for studying the role of a cell edge in vivo and analyzing the effect of competition for a limited amount of tubulin. The model differs from earlier models in that the evolution of MTs is based on the rates of single unit (e.g., a heterodimer per protofilament) transformations, in contrast to postulating effective rates/frequencies of larger-scale changes, extracted, e.g., from the length history plots of MTs. Spontaneous GTP hydrolysis with finite rate after polymerization is assumed, and theoretical estimates of an effective catastrophe frequency as well as other parameters characterizing MT length distributions and cap size are derived. We implement a simple cap model which does not include vectorial hydrolysis. We demonstrate that our theoretical predictions, such as steady state concentration of free tubulin, and parameters of MT length distributions, are in agreement with the numerical simulations. The present model establishes a quantitative link between microscopic parameters governing the dynamics of MTs and macroscopic characteristics of MTs in a closed system. Lastly, we use a computational Monte Carlo model to provide an explanation for non-exponential MT length distributions observed in experiments. In particular, we show that appearance of such non-exponential distributions in the experiments can occur because the true steady state has not been reached, and/or due to the presence of a cell edge.Comment: 14 pages, 7 figure

Caveolin-1–eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability

Caveolin-1–mediated inhibition of endothelial nitric oxide synthase signaling is essential for adherens junction integrity and endothelial permeability homeostasis

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.)

ОСОБЕННОСТИ ПАТОМОРФОЗА ПРИ СОНОДИНАМИЧЕСКОЙ ХИМИОТЕРАПИИ НА ЭТАПАХ ЛУЧЕВОГО ЛЕЧЕНИЯ РАКА СЛИЗИСТОЙ ОБОЛОЧКИ ПОЛОСТИ РТА

Abstract.Morphological response of the tumor was investigated after sonodynamic chemotherapy during distal gamma-therapy (DGT) in patients with cancer of the oral cavity. Sixty-one patients were divided into two groups: group 1 (n=31) received DGT and local ultrasound treatment (0,88 MHz±0,33% and I=1,0Вm/cm2) with 5 mg of platidiam  and group 2 (n=30) received only DGT. Biopsies were investigated by standard morphological and immunohistochemical analysis after achieving total dose of radiation 40 Gy. In the main group the mass of stroma increased 1,9 times after starting of treatment and 1,6 times in comparison with the control group, mitotic activity of the cells decreased 3,9 and 1,9 times respectively, and index of parenchymal damage was 2,4 times higher in the main group than in the control. There was fibrosis and focuses of cells with “cells – shadows” with dystrophic changes of the nucleus and cytoplasma.There was a high index of Ki-67 proliferating activity (60-75%) and high apoptotic index (50-65%) of all cell layers.  The rate of Ki-67 and gene p53 decreased 1,3 and 1,5 times respectively after treatment. In specimens of the main group basal layer cells expressed Ki-67 and p53 in 10-15% of area and didn’t differ from normal values. The rate of KI-67 and expression of p53 gene decreased 7.1 and 6.5 after treatment. Sonodynamic chemotherapy contributes to the suppression of the biological aggressiveness of the tumor, provides a much more pronounced antitumor effect compared to the effect of ionizing radiation alone.Исследовали  морфологический  ответ опухоли на воздействие,  включающее проведение  сонодинамической химиотерапии при дистанционной гамматерапии (ДГТ) рака  слизистой  оболочки  полости  рта (РСПР). 61 больной РПСР были разделены  на основную (31 больному  при ДГТ проводилась   локально  на очаг  опухоли 5 мг платидиама  с помощью  ультразвукового  воздействия  частотой  0,88 МГц±0,33%, и I=1,0Вm/см2) и контрольную (30 больным, сопоставимым по клиническим параметрам,  проводилась  только аналогичная ДГТ) группы. До лечения и после планового перерыва на дозе 40 Гр исследовались  биоптаты опухолей по стандартным морфологическим и иммуногистохимическим методикам. В основной группе доля стромы опухоли увеличилась в 1,9 раз, чем до лечения и в 1,6 раз против контроля, митотическая активность клеток снизилась  в 3,9 и 1,9 раз соответственно, а индекс повреждения паренхимы был выше в 2,4 раза чем в контрольной. Определялся  выраженный фиброз,  наблюдались фокусы клеток, представленные  «клетками — тенями», с выраженными дистрофическими изменениями ядра и цитоплазмыВ контрольной группе было отмечено  сохранение  высокого индекса  пролиферации  Ki-67 (60–75%)  и высокий апоптотический индекс (50–65%)  всех клеточных слоев опухоли. Уровень маркера  Ki-67 и экспрессия мутагенного гена р53 по сравнению с исходными данными снизилась лишь в 1,3 и в 1,5 раз соответственно. В образцах опухоли основной группы клетки базального слоя плоского эпителия вокруг опухоли экспрессировали  Ki-67, p53 в 10–15%  площади и не отличались от нормальных показателей. Уровень маркера  Ki-67 и экспрессия мутагенного гена р53 по сравнению с исходными данными снизились в 7,1 и 6,5 раз соответственно. Таким образом,  сонодинамическая химиотерапия способствует подавлению биологической агрессивности опухоли, обеспечивает существенно более выраженный противоопухолевый эффект по сравнению с действием только ионизирующего излучения

SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure