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Microtubules regulate brush border formation.
Tonucci FM, Ferretti A, Almada E, Cribb P, Vena R, Hidalgo F, Favre C, Tyska MJ, Kaverina I, Larocca MC
(2018) J Cell Physiol 233: 1468-1480
MeSH Terms: Actin Cytoskeleton, Animals, Cell Polarity, Centromere, Colon, Dogs, Enterocytes, Epithelial Cells, Humans, Kidney, Madin Darby Canine Kidney Cells, Microtubule-Associated Proteins, Microtubules, Microvilli, Nocodazole, Time Factors, Tubulin Modulators
Show Abstract · Added April 10, 2018
Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
© 2017 Wiley Periodicals, Inc.
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MeSH Terms
Nuclear LC3 Associates with Slowly Diffusing Complexes that Survey the Nucleolus.
Kraft LJ, Manral P, Dowler J, Kenworthy AK
(2016) Traffic 17: 369-99
MeSH Terms: Active Transport, Cell Nucleus, Cell Nucleolus, HeLa Cells, Humans, Microtubule-Associated Proteins, Protein Binding, RNA, Ribosomal Proteins, Tubulin
Show Abstract · Added February 12, 2016
MAP1LC3B (microtubule-associated protein 1 light chain 3, LC3) is a key component of the autophagy pathway, contributing to both cargo selection and autophagosome formation in the cytoplasm. Emerging evidence suggests that nuclear forms of LC3 are also functionally important; however, the mechanisms that facilitate the nuclear targeting and trafficking of LC3 between the nucleus and cytoplasm under steady-state conditions are poorly understood. In this study, we examine how residues known to regulate the interactions between LC3 and other proteins or RNA (F52 L53, R68-R70 and G120) contribute to its nuclear targeting, nucleocytoplasmic transport and association with nucleoli and other nuclear components. We find that residues F52 L53 and R68-70, but not G120, regulate targeting of LC3 to the nucleus, its rates of nucleocytoplasmic transport and the apparent sizes of LC3-associated complexes in the nucleus inferred from fluorescence recovery after photobleaching (FRAP) measurements. We also show that LC3 is enriched in nucleoli and its triple arginine motif is especially important for nucleolar targeting. Finally, we identify a series of candidate nuclear LC3-interacting proteins using mass spectrometry, including MAP1B, tubulin and several 40S ribosomal proteins. These findings suggest LC3 is retained in the nucleus in association with high-molecular weight complexes that continuously scan the nucleolus.
© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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9 MeSH Terms
Contribution of hepatic organic anion-transporting polypeptides to docetaxel uptake and clearance.
Lee HH, Leake BF, Teft W, Tirona RG, Kim RB, Ho RH
(2015) Mol Cancer Ther 14: 994-1003
MeSH Terms: ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Antineoplastic Agents, Biological Transport, Cell Line, Tumor, Docetaxel, Humans, Liver, Liver-Specific Organic Anion Transporter 1, Male, Mice, Mice, Knockout, Organic Anion Transporters, Organic Anion Transporters, Sodium-Independent, Rats, Solute Carrier Organic Anion Transporter Family Member 1B3, Taxoids, Tubulin Modulators
Show Abstract · Added February 25, 2015
The antimicrotubular agent docetaxel is a widely used chemotherapeutic drug for the treatment of multiple solid tumors and is predominantly dependent on hepatic disposition. In this study, we evaluated drug uptake transporters capable of transporting radiolabeled docetaxel. By screening an array of drug uptake transporters in HeLa cells using a recombinant vaccinia-based method, five organic anion-transporting polypeptides (OATP) capable of docetaxel uptake were identified: OATP1A2, OATP1B1, OATP1B3, OATP1C1, and Oatp1b2. Kinetic analysis of docetaxel transport revealed similar kinetic parameters among hepatic OATP1B/1b transporters. An assessment of polymorphisms (SNPs) in SLCO1B1 and SLCO1B3 revealed that a number of OATP1B1 and OATP1B3 variants were associated with impaired docetaxel transport. A Transwell-based vectorial transport assay using MDCKII stable cells showed that docetaxel was transported significantly into the apical compartment of double-transfected (MDCKII-OATP1B1/MDR1 and MDCKII-OATP1B3/MDR1) cells compared with single-transfected (MDCKII-OATP1B1 and MDCKII-OATP1B3) cells (P < 0.05) or control (MDCKII-Co) cells (P < 0.001). In vivo docetaxel transport studies in Slco1b2(-/-) mice showed approximately >5.5-fold higher plasma concentrations (P < 0.01) and approximately 3-fold decreased liver-to-plasma ratio (P < 0.05) of docetaxel compared with wild-type (WT) mice. The plasma clearance of docetaxel in Slco1b2(-/-) mice was 83% lower than WT mice (P < 0.05). In conclusion, this study demonstrates the important roles of OATP1B transporters to the hepatic disposition and clearance of docetaxel, and supporting roles of these transporters for docetaxel pharmacokinetics.
©2015 American Association for Cancer Research.
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18 MeSH Terms
Microtubule segment stabilization by RASSF1A is required for proper microtubule dynamics and Golgi integrity.
Arnette C, Efimova N, Zhu X, Clark GJ, Kaverina I
(2014) Mol Biol Cell 25: 800-10
MeSH Terms: Apoptosis, Cell Division, Cell Line, Transformed, Cell Polarity, Epithelial Cells, Gene Expression, Golgi Apparatus, Humans, Microtubules, Nocodazole, RNA, Small Interfering, Retinal Pigment Epithelium, Time-Lapse Imaging, Tubulin, Tubulin Modulators, Tumor Suppressor Proteins
Show Abstract · Added March 20, 2014
The tumor suppressor and microtubule-associated protein Ras association domain family 1A (RASSF1A) has a major effect on many cellular processes, such as cell cycle progression and apoptosis. RASSF1A expression is frequently silenced in cancer and is associated with increased metastasis. Therefore we tested the hypothesis that RASSF1A regulates microtubule organization and dynamics in interphase cells, as well as its effect on Golgi integrity and cell polarity. Our results show that RASSF1A uses a unique microtubule-binding pattern to promote site-specific microtubule rescues, and loss of RASSF1A leads to decreased microtubule stability. Furthermore, RASSF1A-associated stable microtubule segments are necessary to prevent Golgi fragmentation and dispersal in cancer cells and maintain a polarized cell front. These results indicate that RASSF1A is a key regulator in the fine tuning of microtubule dynamics in interphase cells and proper Golgi organization and cell polarity.
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16 MeSH Terms
Ice recovery assay for detection of Golgi-derived microtubules.
Grimaldi AD, Fomicheva M, Kaverina I
(2013) Methods Cell Biol 118: 401-15
MeSH Terms: Biological Transport, Cell Line, Centrosome, Golgi Apparatus, Humans, Ice, Microscopy, Confocal, Microscopy, Fluorescence, Microtubule-Organizing Center, Microtubules, Tubulin
Show Abstract · Added March 20, 2014
Proper organization of the microtubule cytoskeleton is essential for many cellular processes including maintenance of Golgi organization and cell polarity. Traditionally, the centrosome is considered to be the major microtubule organizing center (MTOC) of the cell; however, microtubule nucleation can also occur through centrosome-independent mechanisms. Recently, the Golgi has been described as an additional, centrosome-independent, MTOC with distinct cellular functions. Golgi-derived microtubules contribute to the formation of an asymmetric microtubule network, control Golgi organization, and support polarized trafficking and directed migration in motile cells. In this chapter, we present an assay using recovery from ice treatment to evaluate the potential of the Golgi, or other MTOCs, to nucleate microtubules. This technique allows for clear separation of distinct MTOCs and observation of newly nucleated microtubules at these locations, which are normally obscured by the dense microtubule network present at steady-state conditions. This type of analysis is important for discovery and characterization of noncentrosomal MTOCs and, ultimately, understanding of their unique cellular functions.
Copyright © 2013 Elsevier Inc. All rights reserved.
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11 MeSH Terms
Microtubule-regulating kinesins.
Sturgill EG, Ohi R
(2013) Curr Biol 23: R946-8
MeSH Terms: Eukaryota, Humans, Kinesin, Microtubules, Tubulin, Yeasts
Added March 7, 2014
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6 MeSH Terms
GSK3-mediated instability of tubulin polymers is responsible for the failure of immature CD4+CD8+ thymocytes to polarize their MTOC in response to TCR stimulation.
Cunningham NR, Hinchcliff EM, Kutyavin VI, Beck T, Reid WA, Punt JA
(2011) Int Immunol 23: 693-700
MeSH Terms: Aminophenols, Animals, Blotting, Western, CD4 Antigens, CD8 Antigens, Cell Differentiation, Enzyme Inhibitors, Female, Flow Cytometry, Gene Expression Regulation, Developmental, Glycogen Synthase Kinase 3, Lymphocyte Activation, Maleimides, Mice, Mice, Inbred C57BL, Microtubule-Organizing Center, Microtubules, Polymerization, Receptors, Antigen, T-Cell, Signal Transduction, T-Lymphocytes, Thymocytes, Tubulin
Show Abstract · Added January 11, 2016
Although mature T cells divide and differentiate when they receive strong TCR stimulation, most immature CD4+CD8+ thymocytes die. The molecular basis for this marked difference in response is not known. Observations that TCR-stimulated CD4+CD8+ thymocytes fail to polarize their microtubule-organizing center (MTOC), one of the first events that occurs upon antigen activation of mature T cells, suggests that TCR signaling routes in immature and mature T cells diverge early and upstream of MTOC polarization. To better understand the source of the divergence, we examined the molecular basis for the difference in TCR-mediated MTOC polarization. We show that unstable microtubules are a feature of immature murine CD4+CD8+ thymocytes, which also exhibit higher levels of glycogen synthase kinase 3 (GSK3) activity, a known inhibitor of microtubule stability. Importantly, CD4+CD8+ thymocytes gained the ability to polarize their MTOC in response to TCR signals when GSK3 activity was inhibited. GSK3 inhibition also abrogated TCR-mediated apoptosis of immature thymocytes. Together, our results suggest that a developmentally regulated difference in GSK3 activity has a major influence on immature CD4+CD8+ thymocyte versus mature T-cell responses to TCR stimulation.
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23 MeSH Terms
The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly.
Archuleta TL, Du Y, English CA, Lory S, Lesser C, Ohi MD, Ohi R, Spiller BW
(2011) J Biol Chem 286: 33992-8
MeSH Terms: Animals, Bacterial Outer Membrane Proteins, Cattle, Chlamydia, Chlamydia Infections, Metaphase, Microtubules, Spindle Apparatus, Tubulin, Virulence Factors
Show Abstract · Added March 5, 2014
Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system "plug" proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to αβ-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free αβ-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of αβ-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.
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10 MeSH Terms
Optic neuropathy due to microbead-induced elevated intraocular pressure in the mouse.
Chen H, Wei X, Cho KS, Chen G, Sappington R, Calkins DJ, Chen DF
(2011) Invest Ophthalmol Vis Sci 52: 36-44
MeSH Terms: Animals, Anterior Chamber, Axons, Disease Models, Animal, Injections, Intraocular Pressure, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Microspheres, Ocular Hypertension, Optic Nerve Diseases, Polystyrenes, Retinal Ganglion Cells, Time Factors, Tonometry, Ocular, Tubulin
Show Abstract · Added May 28, 2014
PURPOSE - To characterize a glaucoma model of mice, the authors adopted and modified a method of inducing the chronic elevation of intraocular pressure (IOP) by anterior chamber injection of polystyrene microbeads.
METHODS - Chronic elevation of IOP was induced unilaterally in adult C57BL/6J mice by injecting polystyrene microbeads to the anterior chamber. Effectiveness of microbeads of different sizes (small, 10 μm; large, 15 μm) on inducing IOP elevation was compared, and IOP was measured every other day using a tonometer. After maintaining elevated IOP for 2, 4, or 8 weeks, the degree of RGC and axon degeneration was assessed quantitatively using electron microscopy, fluorogold, retrograde labeling, and immunohistochemistry.
RESULTS - Eighty-one of 87 mice that received anterior chamber injection of microbeads exhibited consistent IOP elevation above that of control eyes. Injection of small microbeads induced longer and higher peak value of IOP elevation compared with that of the large microbeads. A single injection of small microbeads resulted in a 4-week elevation of IOP that was maintained to an 8-week period after a second injection of microbeads in week 4. As the duration of IOP elevation increased, RGC bodies and their axons degenerated progressively and reached an approximately 50% loss after an 8-week elevation of IOP.
CONCLUSIONS - Anterior chamber injection of microbeads effectively induced IOP elevation and glaucomatous optic neuropathy in mice. Development of an inducible mouse model of elevated IOP will allow applications of mouse genetic technology to the investigation of the mechanisms and the evaluation of treatment strategies of glaucoma.
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17 MeSH Terms
A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells.
Ferrell N, Desai RR, Fleischman AJ, Roy S, Humes HD, Fissell WH
(2010) Biotechnol Bioeng 107: 707-16
MeSH Terms: Animals, Bioreactors, Cell Proliferation, Dogs, Electric Impedance, Electrodes, Epithelial Cells, Fluorescent Antibody Technique, Humans, Membrane Proteins, Microfluidic Analytical Techniques, Phosphoproteins, Staining and Labeling, Tight Junctions, Tubulin, Zonula Occludens-1 Protein
Show Abstract · Added August 21, 2013
We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell-cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated α-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca(2+) medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca(2+) switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes.
© 2010 Wiley Periodicals, Inc.
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16 MeSH Terms